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TOXIC TRACE METAL REMOVAL USING BIOGENIC MANGANESE OXIDE

IN A PACKED-BED BIOREACTOR

A Master’s Thesis Presented to the Faculty of California Polytechnic State University

San Luis Obispo, California

In partial fulfillment of the requirements for the degree of

Master of Science in General Engineering

with a specialization in Biochemical Engineering

By Jared S. Ervin

March 1, 2005

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COPYRIGHT OF MASTER’S THESIS

I hereby grant permission for the reproduction of this thesis in its entirety or any of its parts, without further authorization, provided acknowledgement is made to the author(s) and advisor(s). Jared S. Ervin ____________________________ Date: ________________________

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MASTER’S THESIS APPROVAL

TITLE: TOXIC TRACE METAL REMOVAL USING BIOGENIC MANGANESE OXIDE IN A PACKED-BED BIOREACTOR AUTHOR: JARED S. ERVIN DATE SUBMITTED: MARCH 1, 2005 THESIS COMMITTEE MEMBERS: Dr. Daniel Walsh ____________________________ Date: ____________________ Dr. Nirupam Pal ____________________________ Date: ____________________ Dr. Yarrow Nelson ____________________________ Date: ____________________

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ABSTRACT

TOXIC TRACE METAL REMOVAL USING BIOGENIC MANGANESE OXIDE IN A PACKED-BED BIOREACTOR

JARED S. ERVIN

The ability of biogenic manganese oxide biofilms to adsorb toxic lead was tested

using a packed-bed bioreactor. Pure cultures of the manganese-oxidizing bacterium

Leptothrix discophora SS-1 were grown and used to create biofilms in two bioreactors

packed with one-quarter inch solid polypropylene beads. Inoculated media trickled down

over the bed like a trickling filter during a growth period of two to three weeks. During

this time, temperature and pH were monitored and kept at approximately 28oC and 7.0,

respectively. After the growth period manganese was added to one of the bioreactors and

the other remained as a control. The bioreactors continued to run for another day to let

the bacterial oxidation of manganese take place. The bioreactors were then flushed, and a

solution of 2 umol/L (414 ppb) lead was run through the columns at 5 mL/min up-flow.

A 10 mL sample of the effluent flow was collected every half hour, equal to one retention

time, for 25 samples. This entire process was repeated three different times with some

alterations in an attempt to maximize lead adsorption to the biofilms. Graphite furnace

atomic absorption spectroscopy (GFAAS) was used to determine the concentration of

lead in the effluent samples. GFAAS was also used to determine the concentration of

manganese in the bed, and the mass of the biofilms was found gravimetrically.

Atomic absorption results from the samples showed that there was lead adsorption

to the oxidized manganese biofilm, but that there was no period of high removal

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efficiency in the beginning of the breakthrough curve. In all experiments the effluent

lead concentration neared that of the influent concentration after just 5 hours (10

retention times) or less. Oxidized manganese deposits on the bioreactor bed were about

0.4 mg and total biomass was about 0.1g. These low levels could account for the low

rates of lead adsorption observed. Results were compared to other researcher’s

adsorption capacities, and it was found that the manganese oxides in this study adsorbed

two orders of magnitude less lead than that reported by another research group. A mass

transfer analysis was also performed which showed that adsorption in the packed bed was

not likely to be limited by the diffusion of lead to the surface of the biofilms on the beads.

Ultimately, the limitation was therefore presumed to be one of the kinetics of lead

adsorption to the biogenic manganese oxide biofilm.

Further experiments should be conducted with a much slower flow rate or more

time given for adsorption to occur. A larger or longer column could also increase

adsorption, and a more tightly packed bed may assist in the growth phase and allow for

greater biomass and in turn higher concentrations of oxidized manganese on the bed.

Further efforts could also be made to establish growth of a pure Leptothrix discophora

SS-1 biofilm. After attaining a successful filter, a commercial product could be produced

that would filter lead and other toxic trace metals out of wastewater and other aqueous

environments.

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ACKNOWLEDGEMENTS

Special thanks to:

Dr. Yarrow Nelson

Dr. Nirupam Pal

Dr. Dan Walsh

Carolyn Zeiner at Cornell

Bunkim Chokshi and Lynne Maloney

All my friends and family

& Christina

This project was supported in part by the Cal Poly C3RP program

through the U.S. Office of Naval Research

For Mom and Dad

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TABLE OF CONTENTS

LIST OF TABLES ………………………………………………………………… ……. x LIST OF FIGURES …………………………………………………………………….. xi INTRODUCTION……………………………………………………………………….. 1 PROJECT SCOPE ………………………………………………………………………. 3 BACKGROUND ………………………………………………………………………... 5

3.1 Biological Manganese Oxidation ………………………………………………... 7

3.1.1 Effects of Condition Changes on Manganese Oxidation …………………. 7

3.1.2 Rate Law for Biological Manganese Oxidation …………………………... 9

3.1.3 Comparison of Biological Manganese Oxidation to Abiotic

Oxidation Rates ………………………………………………………….. 10

3.2 Trace Metal Adsorption to Biogenic Manganese Oxides ……………………… 10

3.2.1 Lead Adsorption to Leptothrix discophora manganese oxides ………….. 11 3.2.2 Discussion of Increased Adsorption …………………………………….. 12

3.3 Manganese Oxide Biofilms …………………………………………………….. 13 3.4 Background Conclusion .……………………………………………………….. 15

MATERIALS AND METHODS……………………………………………………….. 16

4.1 Leptothrix discophora SS-1 Growth …………………………………………… 16 4.2 Bioreactor Design and Construction …………………………………………… 23

4.3 Bioreactor Operation …………………………………………………………… 28

4.3.1 Biofilm Growth ………………………………………………………….. 29 4.3.2 Manganese Oxidation …………………………………………………… 30

4.4 Lead Adsorption ………………………………………………………………... 31

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4.5 Atomic Absorption Spectroscopy ……………………………………………… 32

4.5.1 Lead Analysis by Atomic Absorption …………………………………… 33

4.5.2 Manganese Analysis by Atomic Absorption ……………………………. 35

4.6 Biomass Characterization ……………………………………………………… 37

4.6.1 Microscopy ……………………………………………………………… 38 4.6.2 Dry Weight Analysis …………………………………………………….. 38

RESULTS ……………………………………………………………………………… 40

5.1 Results from First Complete Run ……………………………………………… 40

5.1.1 Lead Breakthrough Curve for First Run ………………………………… 41 5.1.2 Manganese Analysis for First Run ………………………………………. 45

5.1.3 Biomass Dry Weight for First Run .……………………………………... 47

5.1.4 Observations and Microscopy for First Run …………………………….. 48

5.2 Results from Second Complete Run …………………………………………… 50

5.2.1 Lead Breakthrough Curve for Second Run …….………………………... 50 5.2.2 Manganese Analysis for Second Run …………………………………… 53

5.2.3 Biomass Dry Weight for Second Run …………………………………… 55

5.2.4 Observations and Microscopy for Second Run …………………………. 56

5.3 Results from Third Complete Run ………………………………....................... 58

5.3.1 Lead Breakthrough Curve for Third Run ………………………………... 58 5.3.2 Manganese Analysis for Third Run ……………………………………... 61

5.3.3 Biomass Dry Weight for Third Run ……………………………………... 63

5.3.4 Observations and Microscopy for Third Run …………………………… 64

5.4 Lead Removal Efficiency for All 3 Runs ……………………………………… 66

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DISCUSSION ………………………………………………………………………….. 68

6.1 Lead Adsorption Results Discussion …………………………………………... 68 6.2 Comparison to Lead Adsorption by Manganese Oxides in Other Studies …...... 73

6.2.1 Quantity of Manganese in the Biofilms …………………………………. 73 6.2.2 Quantity of Lead Adsorbed to the Biofilms ……………………………... 74

6.3 Mass Transfer Analysis…………………………………………………………. 75

6.4 Possible Future Experiments …………………………………………………… 77

6.5 Potential Applications ……………………………….………………................. 78

CONCLUSIONS ………………………………………………………………………. 79

7.1 Summary ……………………………………………………………………….. 79 7.2 Future Recommendations ……………………………………………………… 81

REFERENCES ………………………………………………………………………… 83

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LIST OF TABLES

Table 1 Composition of Pyruvate Growth Media …………………………......... 17 Table 2 Composition of MMS Growth Media ………………………………….. 22 Table 3 Results from Lead Standards by GFAAS from 8/17/2004 …………….. 41 Table 4 Results for Lead Samples from Column #1, Manganese Added, by

GFAAS from 8/17/2004 ……………………………………………….. 43 Table 5 Results for Lead Samples from Column #2, Control with No Manganese

Added, by GFAAS from 8/17/2004 ……………………………………. 44 Table 6 Results from Manganese Standards by GFAAS from 8/17/2004 ……… 46 Table 7 Biomass Dry Weight from 8/17/2004 ………………………………….. 47

Table 8 Results from Lead Standards by GFAAS from 11/6/2004 …………….. 51 Table 9 Effluent Lead Concentrations from Column #1, Manganese Added, and

Column #2, Control with No Manganese Added, by GFAAS from 11/6/2004 ………………………………………………………………. 52

Table 10 Results from Manganese Standards by Graphite Furnace Atomic

Absorption Spectroscopy from 11/6/2004 ……………………………... 54 Table 11 Biomass Dry Weight from 11/6/2004 ………………………………….. 55

Table 12 Results from Lead Standards by GFAAS from 12/3/2004 …………….. 59 Table 13 Results for Lead Samples from Column #1 and Column #2, Both with

Manganese Added, by GFAAS from 12/3/2004…………………........... 60 Table 14 Results of Manganese Concentration on the Column Beds from

12/3/2004 ………………………………………………………………. 62 Table 15 Biomass Dry Weights from 12/3/2004……………………………......... 63 Table 16 Total Biomass and Manganese Results from All Complete Runs ……... 72

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LIST OF FIGURES

Figure 1 Plate Streaking Procedure ……………………………………………… 19 Figure 2 Reservoir Design ……………………………………………………….. 24

Figure 3 Packed-bed biofilm Column Design …………………………………… 26

Figure 4 Bioreactor Apparatus …………………………………………………... 27

Figure 5 Up-flow Lead Adsorption Column …………………………………….. 32

Figure 6 Lead Graphite Furnace Atomic Absorption Conditions ……………….. 35

Figure 7 Manganese Graphite Furnace Atomic Absorption Conditions ………… 37

Figure 8 Lead Calibration Curve by GFAAS from 8/17/2004 …………………... 42

Figure 9 Breakthrough Curves for L. discophora Lead Adsorption Over Time by Both Control and Sample Column Packed-beds With and Without Manganese Oxide from 8/17/2004 ……………………………………... 45

Figure 10 Manganese Calibration Curve by GFAAS from 8/17/2004 …………… 46 Figure 11 1000X Magnification of Media Sample Taken from the Sample Column

on 8/17/2004 …………………………………………………………… 48 Figure 12 1000X Magnification of Media Sample Taken from the Control Column

on 8/17/2004 …………………………………………………………… 49 Figure 13 Lead Calibration Curve by GFAAS from 11/6/2004 …………………... 51 Figure 14 Breakthrough Curves for L. discophora Lead Adsorption Over Time by

Both Control and Sample Column Packed-beds With and Without Manganese Oxide from 11/6/2004 ……………………………………... 53

Figure 15 Manganese Calibration Curve by GFAAS from 11/6/2004 …………… 54 Figure 16 1000X Magnification of Media Sample Taken from the Sample Column

from 11/6/2004 …………………………………………………………. 56 Figure 17 1000X Magnification of Media Sample Taken from the Control Column

from 11/6/2004 …………………………………………………………. 57 Figure 18 Lead Calibration Curve by GFAAS from 12/3/2004 …………………... 59

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Figure 19 Lead Breakthrough Curves for L. discophora Lead Adsorption Over Time by Both Packed-bed Columns With Manganese Oxide from 12/3/2004 ………………………………………………………………. 61

Figure 20 1000X Magnification of Media Sample Taken from the Sample Column

#1 from 12/3/2004 ……………………………………………………… 64 Figure 21 1000X Magnification of Media Sample Taken from the Sample Column

#2 from 12/3/2004 ……………………………………………………… 65 Figure 22 Lead Adsorption Results from All 3 Runs ……………………………... 66 Figure 23 1000X Magnification of pure L. discophora from Inoculation Broth …. 71 Figure 24 1000X Magnification of pure L. discophora from Growth Plate with

Oxidized Manganese Present …………………………………………... 71

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CHAPTER 1

INTRODUCTION

Toxic trace metals, such as lead, can be hazardous even at very low

concentrations (Nriagu, 1990). When they get into water supplies and aqueous

environments the health of plants and animals, as well as humans, can be impaired.

Toxic trace metals are commonly found in wastewater and removing them efficiently

presents a unique challenge. Whether contamination occurs as a result of human

intervention or naturally, an easy and non-intrusive way of cleaning up such hazards

would be beneficial to everyone. In the environment biologically formed manganese and

iron oxides regulate the bioavailability of some toxic trace metals (Nelson et al., 1999a).

Metals are bound to the biogenic manganese and iron oxides and removed from solution

rendering the solution free of toxic trace metals. This adsorption process could

potentially be exploited for the development of engineered toxic metal removal

processes.

Previous research has shown that manganese oxide biofilms produced by

Leptothrix discophora SS-1 have a high affinity for the binding of toxic lead from an

aqueous solution (Nelson et al., 1999b). In some cases the metal binding of the biogenic

manganese oxides was orders of magnitude greater than that of abiotic manganese oxide

minerals. This research concluded that further experimentation needed to be done on the

ability of biogenic manganese oxides to bind lead and their possible uses in more

practical engineering applications. This previous research is what prompted

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experimentation on building a laboratory filter that uses a biogenic manganese oxide

biofilm to remove toxic trace metals from an aqueous solution.

This project was designed to test the ability of a biogenic manganese oxide

biofilm to filter toxic lead out of an aqueous solution using a packed-bed bioreactor.

Biofilms of L. discophora were grown on plastic beads and manganese was added to

allow for the formation of biogenic manganese oxides on the surfaces of the beads.

Aqueous lead solutions were then pumped through the packed-bed columns. Atomic

absorption spectroscopy was used to determine lead concentrations in filtered solutions

and manganese concentrations on the bioreactor bed. Details on the scope of this project

are presented in the next chapter. Results from this project will give insight into what

further steps need to be taken in order to further this technology. It will also help

determine the real world effectiveness of such a filter, possible engineering applications,

and the feasibility of producing a commercial product at some point in the future.

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CHAPTER 2

PROJECT SCOPE

Laboratory experiments were conducted on the ability of biogenically oxidized

manganese biofilms to filter toxic lead in a packed-bed bioreactor. Two packed-bed

bioreactors were designed and built to conduct the experiments in. Two bioreactors

allowed a control to be run simultaneously in each experiment. The bioreactors were

packed with one-quarter inch solid polypropylene balls to facilitate a large surface area

on which to grow the biofilms. Pure cultures of Leptothrix discophora SS-1 were grown

and used to create biofilms in the bioreactors by trickling inoculated media down over the

bioreactor beds. The trickling filter design allowed for maximum aeration and nutrients

to be brought to the biofilm from a reservoir at the same time. The biofilms were given

two to three weeks to grow in each experiment before manganese was added. The

control was kept free of manganese in order to determine the adsorption effects of the

bioreactors without oxidized manganese. Manganese sulfate solution was recirculated

through the bioreactor for one day to allow for manganese oxidation. Finally, the

bioreactor beds were flushed with a media solution and a solution of lead was run up-

flow through the bioreactor beds. Up-flow allowed for a low flow rate that was thought

to be necessary to get good binding of the lead to the oxidized manganese biofilm.

Samples of the effluent were collected and analyzed for lead concentrations using

GFAAS. The graphite furnace was necessary in order to detect the low levels of lead

present in the samples. The bioreactor bed was then dried and weighed before dissolving

the biofilm and oxidized manganese in nitric acid. GFAAS was then used to determine

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the concentration of manganese on the bioreactor bed. This information along with the

dry weight of the biofilm was gathered in order to characterize the biomass. Three

complete experimental processes were completed over the course of one year with slight

variations to each experiment in an attempt to maximize lead adsorption.

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CHAPTER 3

BACKGROUND

This project was based on the formation of biogenic manganese oxides in a

biofilm and their ability to adsorb toxic trace metals. Previous research into this subject

has been done, but much is still unknown about the subject. It has been recognized that

in the environment biological manganese oxidation is important in controlling not only

the bioavailability of manganese, but also the bioavailability of other trace metals

(Nelson et al., 1999a). This includes toxic trace metals such as lead that are strongly

bound to suspended biogenic manganese oxides. Manganese cycling is very important in

the environment, and research has been done into the enzymatic pathways responsible for

biological manganese oxidation (Tebo et al., 1997). Other research has also been done

into a kinetic model for biological manganese oxidation (Zhang et al., 2002) and the

effects of strong trace metal binding by biogenic manganese oxides in aqueous

environments (Nelson et al., 1999a; Nelson et al., 2003; Dong et al., 2003). This research

has given insight into biogenic manganese oxidation and spurred the need for more

research to be completed to further understand the subject.

Manganese oxidation can occur without the help of microorganisms, but abiotic

manganese oxidation occurs only at a high pH and is therefore not expected to frequently

occur in nature (Tebo et al., 1997). This means that manganese oxidation in the

environment is mostly due to biological processes. There are two theories of how this

might occur. Biological manganese oxidation could occur as the result of an

enzymatically catalyzed reaction or as the result of local changes in pH caused by other

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microorganisms such as algae (Nelson et al., 2003). There are several microorganisms

with the reported ability to biologically oxidize manganese that have been found and

isolated in the environment. The bacterium Bacillus subtilis, found in the ocean, the

bacterium Pseudomonas putida MnB1, found in freshwater, and the bacterium that was

used in this project, Leptothrix discophora, which was isolated from the metallic surface

film of freshwater wetlands in New York State (ATCC, 2004), all have the ability to

biologically oxidize manganese.

This chapter will focus on several different aspects of biological manganese

oxidation in order to gain a complete understanding of the subject and the work that has

been done on it. The first section will focus on biological manganese oxidation and the

kinetics that follow. Here the research that led up to the creation of a rate law for

biologically catalyzed manganese oxidation will be discussed. Next, the focus will shift

to the ability of those biological manganese oxides to adsorb trace metals and the

mechanism by which they do this. And finally, there will be a discussion of the research

that has been done on trace metal adsorption to natural biofilms that contain biogenic

manganese oxides. Researchers have long thought that biologically formed manganese

oxides would have a much higher trace metal binding capability than those formed

abiotically and recent research done to find out if that is true will be discussed. That will

then conclude the bulk of the literature review that is pertinent to the understanding of

and prompting of this project.

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3.1 Biological Manganese Oxidation

When manganese oxidation was first studied it was only abiotic methods that

were taken into account and biological manganese oxidation was completely ignored.

However, abiotic manganese oxidation occurs readily only at a pH of 9 or greater and

proceeds extremely slowly at lower pH’s like those found in the natural environment

(Nelson et al., 2003). Because of this it is generally recognized that in order for the

reaction to occur there must be some kind of biological catalyst. It has been shown that

manganese oxidation in a natural environment at a pH near neutral can occur at a rate

several orders of magnitude greater than that of abiotic manganese oxidation (Nealson et

al., 1988). From this research it was clear that a kinetic model for the biogenic oxidation

of manganese at neutral pH levels needed to be developed. Reported rates of biological

manganese oxidation vary greatly depending on growth medium, conditions, and

bacterial strain used in each experiment. Temperature and pH also play a major role in

the rate of biological manganese oxidation in natural environments as well as in pure

cultures of Leptothrix discophora SS-1. It has been shown that Leptothrix discophora

SS-1 exhibits a maximum rate of manganese oxidation at a pH of 7.5 and a temperature

of 30oC (Adams and Ghiorse, 1987).

3.1.1 Effects of Condition Changes on Manganese Oxidation

Experiments were conducted over a range of conditions using the bacterium

Leptothrix discophora SS-1 (Nelson et al., 2003). In one experiment variables included

temperature, pH, and the concentrations of cells, manganese, oxygen and copper. As in

previous research, biological manganese oxide biofilms were grown in laboratory

controlled bioreactors and in a defined growth medium (Zhang et al., 2002). A defined

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growth media is necessary when doing this research because undefined media ingredients

could contain trace metals and some buffers could interfere with manganese oxidation.

The results of this research showed that biological manganese oxidation was directly

proportional to cell and oxygen concentrations and that there was a pH optimum of 7.5

and a temperature optimum of 30oC, which was consistent with previous research

(Adams and Ghiorse, 1987). It was also found that for Leptothrix discophora SS-1

manganese oxidation kinetics were consistent with Michaelis-Menton enzyme kinetics in

terms of manganese concentration (Nelson et al., 2003; Tebo and Emerson, 1986).

Michaelis-Menton parameters were then determined for Leptothrix discophora SS-1

(Zhang et al. 2002). At optimum conditions, the maximum biological manganese

oxidation rate was determined to be 0.0059 umol Mn/min-mg cell, and the half velocity

coefficient for biological manganese oxidation by Leptothrix discophora SS-1 was

determined to be 5.7 umol Mn/L (Zhang et al., 2002). These Michaelis-Menton

parameters were used in the development of a rate law for the biological oxidation of

manganese by Leptothrix discophora SS-1 under these ideal conditions.

Other research has shown that copper concentrations are important in the

biological oxidation of manganese (Corstjens et al., 1992). Studies looking into the

molecular biology of manganese oxidizing bacteria have shown that enzymes containing

copper may play an important role in bacterial manganese oxidation (Corstjen et al.,

1992). Addition of copper at concentrations as low as 0.02 uM has been shown to

increase the rate of manganese oxidation by 25%, but at the same time slightly inhibit the

growth rate and ultimate biomass yield when using Leptothrix discophora SS-1 (Zhang et

al., 2002). Zhang et al. also showed that it was important for copper to be present in the

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growth media from the beginning. When copper was added after growth was complete it

did not increase the rate of manganese oxidation.

3.1.2 Rate Law for Biological Manganese Oxidation

With information on the effects of the parameters described above it was then

possible to develop a general rate law for biological manganese oxidation by Leptothrix

discophora SS-1. The rate law took into account the effects of cell concentration,

manganese concentration, pH, temperature, dissolved oxygen concentration, and copper

concentration and is shown in Equation 1 (Zhang et al., 2002).

)])([1(]/[/][1

)])([()]([)](][[)]([

21

/22 IICuk

HKKHk

AeOkIIMnKIIMnXk

dtIIMnd

cpHRTEa

os

+⎟⎟⎠

⎞⎜⎜⎝

⎛+++

=− ++− (Eq. 1)

In Equation 1, [X] = cell concentration, mg/L; [O2] = dissolved oxygen

concentration, mg/L; [Cu] = total dissolved copper concentration, umol/L; k = 0.0059

umol Mn(II)/(mg cell · min); Ks = 5.7 umol Mn(II)/L; ko2 = 1/8.05 = 0.124 L/mg ([O2] =

8.05 mg/L at 25oC and I = 0.05 mol/L); Ea = 22.9 kcal/(g cell · mol); A = 2.3 × 1014; K1 =

3.05 × 10-8; K2 = 2.46 × 10-8; kpH = 2.82; kC = 8.8 L/umol Cu. Under typical conditions,

temperature = 25 oC, pH = 7.5, [O2] = 8.05 mg/L and zero added copper, the above

equation can be simplified into Equation 2, which is the Michaelis-Menton expression for

biological manganese oxidation.

)]([)](][[)]([

IIMnKIIMnXk

dtIIMnd

s +=− (Eq. 2)

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3.1.3 Comparison of Biological Manganese Oxidation to Abiotic Oxidation Rates

The rate law described above was then used to compare the rate of biological

manganese oxidation to that of abiotic manganese oxidation (Nelson et al., 2003). It was

found that at a pH of 8.03 it would only take a Leptothrix discophora SS-1 cell

concentration of 0.30 ug/L to match the rate of abiotic manganese oxidation at the same

pH. Cell populations of manganese oxidizing bacteria much higher than this can be

found in natural environments, and an even smaller concentration of manganese

oxidizing bacteria would be necessary to match the abiotic rate at a lower pH. This rate

law equation is only true for Leptothrix discophora SS-1 and other bacteria or even other

strains of Leptothrix discophora would almost certainly exhibit different rates of

biological manganese oxidation. However, the general form of the equation would most

likely be the same for other manganese oxidizing bacteria and other strains of Leptothrix

discophora. This research helped to improve validity of the theory that manganese

oxidation in natural environments is controlled by manganese oxidizing bacteria and not

abiotic reactions.

3.2 Trace Metal Adsorption to Biogenic Manganese Oxides

The most crucial research that led up to this project was that of toxic trace metal

adsorption by biogenic manganese oxides. It has been shown that in natural

environments toxic trace metals are controlled by interactive biogeochemical processes

including adsorption, complexation and multiple biological interactions (Nelson et al.,

1999a). Previous research has also shown that microorganisms have the ability to adsorb

large amounts of toxic trace metals and many bacteria are able to bind metals to

extracellular polymers that they produce (Lion et al., 1988; Nelson et al., 1995). Certain

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trace metals, however, have a much higher potential to be adsorbed to biogenic

manganese and iron oxides than to be adsorbed by organic material. The next section

will focus on research done on the binding capabilities of trace metals to biogenic

manganese oxides in controlled laboratory conditions.

3.2.1 Lead Adsorption to Leptothrix discophora Manganese Oxides

The only significant research to date into trace metal adsorption by biologically

formed manganese oxides was done by Nelson et al. (2003). Manganese oxidation and

trace metal adsorption experiments were conducted under controlled laboratory

conditions using Leptothrix discophora as the manganese oxidizing bacteria in a

chemically defined growth medium. Because buffers can complex manganese they were

left out of the growth medium, and pH controllers were used to control the pH. Other

trace metals were also omitted from the growth medium except for a very small amount

of iron (0.1 uM), which was found to be necessary for manganese oxidation (Nelson et

al., 1999b). In this research it was found that at a pH of 6.0 and a temperature of 25 oC,

lead adsorption by Leptothrix discophora cells with biogenic manganese oxide coatings

was two orders of magnitude greater than lead adsorption by the same cells without

biogenic manganese oxide coatings (Nelson et al., 1999b). This confirmed the theory

that lead has a much higher binding affinity for metal oxides than it does for organic

material. However, the research also found that lead had a much higher binding affinity

for biogenic manganese oxides than for abiotic manganese oxides. Experiments

conducted under the same conditions as above showed that biogenic manganese oxides

had five times the lead adsorption capacity as that of freshly prepared abiotic manganese

oxides (Nelson et al., 1999b). Other results from the same research showed that the

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difference in lead adsorption by biogenic manganese oxides compared to abiotic

manganese oxides was even more significant at very low lead concentrations like those

that would be found in natural aquatic environments. It was also found that adsorption of

lead by biogenic manganese oxides under the same conditions was several orders of

magnitude greater than that of abiotic pyrolusite manganese oxide minerals and more

than an order of magnitude greater than colloidal iron oxyhydroxide (Nelson et al.,

1999b). This research concluded that even though there is much less manganese than

iron in natural aquatic environments, the increased ability of manganese oxides to absorb

lead may make manganese oxides as important or even more important than iron oxides

in the natural adsorption of some toxic trace metals (Nelson et al., 1999b).

3.2.2 Discussion of Increased Adsorption

Further research was conducted into why biogenic manganese oxides have such

an increased affinity for the binding of lead and some other toxic trace metals.

Research was conducted on biogenic manganese oxides formed in the same controlled

laboratory conditions as above using x-ray diffraction analysis (Nelson et al., 2001). The

analysis showed that the biogenic manganese oxides were amorphous and would thus be

expected to exhibit greater surface area than other types of manganese oxide examined.

The specific surface area of biogenic manganese oxide was found to be 220 m2/g (Nelson

et al., 2001). This research found that the greater surface area was proportional to the

greater lead binding affinity in biogenic manganese oxides over abiotic manganese

oxides. However, this relationship did not hold true when comparing biogenic and

freshly precipitated abiotic manganese oxides to crystalline pyrolusite manganese oxides.

The ratio of lead adsorption to surface area was much greater for the biogenic and abiotic

13

manganese oxides (Nelson et al., 2003). Therefore, it was concluded that surface area

may play an important role in the increased lead adsorption of manganese oxides, but

other surface properties of the highly adsorbent biogenic manganese oxides may also be

important.

The comparison of adsorption by manganese oxides to that of iron oxides depends

on pH because pH strongly affects cation adsorption. When lead adsorption to biogenic

manganese oxides was compared with lead adsorption to colloidal iron oxides over a

range of pH’s, it was shown that biogenic manganese oxides had a higher affinity for

binding lead than iron oxides at pH below 8.5 and iron oxides had a greater affinity at pH

above 8.5 (Nelson et al. 2003). These studies demonstrated the importance of biogenic

manganese oxides in the binding of trace metals in the environment and the need for

further research.

3.3 Manganese Oxide Biofilms

This thesis investigated the ability of biogenic manganese oxide biofilms to

adsorb toxic trace metals and there has been some previous research done on this subject.

In natural aquatic environments, manganese oxidizing bacteria such as Leptothrix

discophora tend to form biofilms (Ghiorse et al., 1984). The manganese oxides are

contained in these biofilms and it is in the biofilm that trace metal adsorption takes place.

Research was done to determine the extent of trace metal adsorption by biogenic

manganese oxide biofilms in natural environments (Nelson et al., 2003). Two methods

were developed in order to determine this. The first method made use of a surrogate

adsorption and additivity model to measure the relative contributions of manganese

oxides, iron oxides and other organic materials in lead adsorption to a natural biofilm

14

(Nelson et al., 1999a). It was determined, as expected, that at a pH of 6.0 manganese

oxides were responsible for the most lead adsorption when experiments were conducted

with natural biofilms from several freshwater lakes (Nelson et al., 1999a). Further

research was conducted to determine lead adsorption at several different pH’s (Wilson et

al., 2001). This research concluded that manganese oxides were responsible for the most

lead adsorption below a pH of 6.5 and iron oxides were most responsible at pH’s above

6.5 with manganese oxides second (Wilson et al., 2001).

The second method made use of selective extraction experiments in which surface

coatings on glass slides from a freshwater lake in New York were tested for lead and

cadmium adsorption (Dong et al., 2000). Adsorption measurements were made after

each extraction to determine the role of the extracted material in lead and cadmium

adsorption. Extractions of manganese oxides, manganese and iron oxides, and

manganese and iron oxides and other organic matter were performed and subsequent lead

and cadmium adsorption were compared for each. As with the first method above, it was

determined that manganese oxides played the largest role in the adsorption of lead with

iron oxides second (Dong et al., 2000). Iron oxides were found to be most dominant in

cadmium adsorption, which shows that different sorbents can dominate adsorption

depending on the trace metal being analyzed. Further research was performed on

manganese and cadmium adsorption under actual lake conditions (Dong et al., 2003).

This research also concluded that manganese oxides were the dominate factor in the

adsorption of lead to natural biofilms.

15

3.4 Background Conclusion

With an understanding of biogenic manganese oxidation, trace metal adsorption

to biogenic manganese oxides and manganese oxide biofilms and the research that has

been performed in these areas it is easy to see why continued research is necessary. The

lead adsorption capabilities of biogenic manganese oxides in the natural environment

have been well documented. There is, however, a need for this phenomenon to be

engineered into something practical. It is this need that prompted this thesis project.

16

CHAPTER 4

MATERIALS AND METHODS

There were five major steps in my research. There was the growth of pure

cultures of manganese oxidizing bacteria, the design and construction of bioreactors, the

operation of those bioreactors, trace metal adsorption in the bioreactors, and finally

analysis of the trace metal adsorption, which included atomic adsorption spectroscopy of

both lead and manganese and characterization of the biomass. The first step in the

project turned out to be one of the most difficult and time consuming. The next section

focuses on the steps taken in the growth of pure cultures of manganese oxidizing bacteria,

and is followed by sections containing details of the other four steps.

4.1 Leptothrix Discophora SS-1 Growth

The bacterial strain Leptothrix discophora SS-1 (L. discophora) was chosen for

this project because of its well documented ability to form biogenic manganese oxide

biofilms and bind lead to them (Nelson et al., 2003). Pure cultures of L. discophora were

ordered from The American Type Culture Collection (ATCC #43182) and received on a

large Petri dish that had been frozen for preservation.

Growth media was prepared in order to transfer and grow pure cultures of L.

discophora. ATCC 1503, the recipe for growth media recommended by the ATCC for L.

discophora, was used with the exception that 0.5 g pyruvate was used instead of 0.5 g

glucose as a carbon source for the bacteria. This switch was made in order to get the

bacteria accustomed to growing on a pyruvate based media, which is the type of media

that would be used later in the experiment. Pyruvate has previously been shown to be a

17

good carbon source for L. discophora (Adams and Ghiorse, 1987). The recipe as used is

shown in Table 1.

Table 1 Composition of Pyruvate Growth Media

Peptone …………….. 0.5 g

Yeast extract ……….. 0.5 g

Pyruvate …………….. 0.5 g

MgSO4 · 7H2O ……… 0.6 g

CaCl2 · 2H2O ……….. 0.07 g

HEPES ……………… 3.57 g

MnSO4 · H2O ……….. 17.0 mg

Distilled Water ……… 1.0 L

Each ingredient in Table 1, except pyruvate, was added to one liter of deionized

(DI) water. Pyruvate was left out because it would break down during the autoclaving

process. Once all the above ingredients were added, 1.5% agar was added (15 g for 1 L)

to the solution to make the media harden after autoclaving for streak plates and slant

tubes. The pH was measured using a pH probe and then adjusted by adding 1% NaOH in

DI water 1 mL at a time until the pH was just above 7.0. After pH adjustment, the media

was poured into a 2 L media bottle. A cap was placed loosely on top of the bottle and

then the whole bottle was autoclaved at 121 oC for 15 minutes. Having 1 L of media in a

2 L bottle allows for some boiling to occur without spilling out of the bottle. After

autoclaving, the cap was tightened and the media was allowed to cool until safe to handle

with bare hands. Pyruvate was then introduced to the media by adding 10 mL of a 50 g/L

pyruvate solution. The 50 g/L pyruvate solution had been prepared by adding 5 g of

pyruvate to 100 mL of DI water. This solution was then filter sterilized with a disposable

18

0.2-micron Nalgene® filter to remove all contaminants, and excess solution was stored in

the refrigerator for later use. After adding the pyruvate, the bottle was shaken to

completely mix up the pyruvate solution. Streak plates were then prepared by pouring

the media into sterile Petri dishes and allowing them to cool and harden. About 20 mL of

media was poured into each Petri dish, the dish was gently swirled for even coverage and

then the sterile lid was placed on the dish. Slant tubes were also prepared by pouring

about 10 mL of media into sterile test tubes and capping them with a sterile lid. The

tubes were then tilted at an angle of about 65 degrees and allowed to harden while tilted.

This angle gives a large slanted surface area for the bacteria to grow on once streaked.

All pouring was done under a sterile laminar flow hood to avoid contamination as much

as possible. Once hardened the plates and slants were stored in a refrigerator for later

use.

The streaking of plates is designed to spread bacteria out on a media surface to

allow pure isolated colonies of the desired bacteria to grow. The process started by

removing a plate with pure isolated colonies of L. discophora and about five empty plates

from the refrigerator and allowing them to warm up a bit. An inoculation loop was

flamed until red hot, allowed to cool for a few seconds and then touched to a single pure

colony of L. discophora. The loop was then touched to an empty streak plate and moved

back and forth across the surface on one third of the plate. The loop was then flamed

again, moved through the area that was just streaked a couple of times and then moved

back and forth across the surface on a second third of the plate. The loop was flamed

again, moved through the second streak a single time and then moved back and forth

across the last third of the plate. This process allows for three dilutions of bacterial

19

concentration to occur by spreading them out so that single bacterium will be separated.

A single pure colony will then be created from that single bacterium. The streaking

process is depicted in Figure 1.

Figure 1 Plate Streaking Procedure

This process was usually repeated on five or more plates in order to ensure plenty

of L. discophora growth and pure isolated colonies. All streaking was performed under a

laminar flow hood and bacteria on the plates were left there to grow at room temperature

for three to five days depending on visible growth. Plates streaked from the dish obtained

from the ATCC showed no L. discophora growth and a new source of bacteria had to be

found. At the time, research was being conducted at Cornell University using L.

discophora SS-1 and they were kind enough to send some bacteria. Plates were streaked

and then allowed to grow while in the mail. This provided freshly grown L. discophora

1

2

3

20

and the bacteria were immediately transferred onto more streak plates by the process

above.

Bacteria were also transferred onto slant tubes from the streak plates. Four or five

empty slant tubes were removed from the refrigerator and allowed to warm up along with

a streak plate with pure isolated colonies of L. discophora. An inoculation loop was

flamed and then touched to a single colony on the streak plate. The loop was then

touched to the bottom of the surface of the media in the slant tube and moved upward

across the surface in a single streak. Slant streaking and growth was also performed

under a laminar flow hood. The slants were then allowed to grow at room temperature

for three to five days depending on visible growth. Slant tubes were used in addition to

streak plates because they are much better for storing bacteria for longer periods of time.

Once visible growth was observed, bacterial colonies on the plates and slants

were observed under the microscope to make sure that they were pure L. discophora. An

Olympus BX50 optical microscope with an Olympus OLY-200 integrated camera, for

capturing digital images, was used for all microscopy performed. 8 uL of DI water was

pipetted onto a clean microscopic slide and then a flamed inoculation loop was touched to

a single colony of bacteria and then to the drop of DI water. A cover slip was then placed

over the drop and the slide was mounted on the microscope. The slide was first brought

into focus at 100X magnification and phase contrast 1 and then switched to 1000X

magnification and phase contrast 3. At this magnification it was easy to see if the

bacteria on the slide were pure L disc. L disc could also be identified directly on the

slants and plates by the brown color of their colonies. The brown color is due to the

manganese in the growth media being oxidized by the bacteria.

21

After streak plates and slant tubes were grown they were placed in the refrigerator

to stop growth and preserve the fresh colonies. Plates and tubes were streaked

approximately every week to ensure that fresh L. discophora were always available.

Bacteria on plates from the week before were streaked onto new slants and bacteria from

slants from the week before were streaked onto new plates to keep the bacteria pure.

Bacterial cultures needed to be in a different media and in broths for the experiment. A

minimal mineral salts (MMS) media was chosen for the broths because it is a defined

media with no unnecessary trace metals or buffers. The recipe for the media is shown in

Table 2. For this media the Vitamin B12 and the FeSO4, in addition to the pyruvate, were

left out of the media until after autoclaving was complete and the solution had cooled

down. The rest of the ingredients were added and the media was prepared, pH balanced

to 7.0, and autoclaved in the same way as the Pyruvate media. Solutions of 2 mg/L

Vitamin B12 and 15 mg/L FeSO4 were made and filter sterilized into separate sterile

bottles so that 1 mL of each could be added to the media once it had cooled down. These

stock solutions were also stored in the refrigerator for later use. Four or five 250-mL

Erlenmeyer flasks with silicone closures were autoclaved along with the media. After

cooling, 100 mL of media was poured into each of the Erlenmeyer flasks. This allowed

for plenty of surface area in the flask to allow good aeration. After pouring the rims of

the Erlenmeyer flasks were flamed before replacing the silicone closures, to prevent any

contamination. Remaining media was stored in the refrigerator for later use. The media

was inoculated by flaming an inoculation loop and touching it to a single pure colony of

L. discophora on a streak plate and then dipping the loop into the media in the

Erlenmeyer flask. Inoculation was done under a laminar flow hood and the flasks were

22

then placed on a shaker table to grow at room temperature for three days to a week,

depending on visible growth. The shaker table was set to 100 rotations per minute to

provide aeration and mixing.

Table 2 Composition of MMS Growth Media

Pyruvate …………… 240 mg

CaCl2 · 2H2O ……… 30 mg

MgSO4 · 7H2O …..... 35 mg

(NH4)2SO4 ………………... 120 mg

KNO3 ………………………… 15 mg

NaHCO3 ………………....... 0.84 mg

KH2PO4 …………………….. 0.70 mg

Vitamin B12 ………………. 0.002 mg

FeSO4 ………………………. 0.015 mg

Distilled Water …..... 1.0 L

Although fresh healthy colonies of L. discophora were used, no growth was

observed in the MMS medium and it was decided that a more nutrient-rich media would

need to be used. 1 L of 1987 growth media was prepared and autoclaved in the same way

as the other medias. 1987 growth media is the same as Pyruvate growth media except

there is no MnSO4 · H2O added. The media needed to be in liquid form so no agar was

added either. Experiments on growth were conducted by adding small amounts of the

1987 growth media to the MMS growth media and inoculating. Flasks with MMS

growth media and 1%, 5%, 10% and 25% 1987 growth media were prepared and all

inoculated at the same time from the same source. After five days of growth the flasks

were examined to compare growth at different 1987 media concentrations. Broths were

also checked under the microscope to make sure there was pure L. discophora growth.

23

No growth was observed in the flask with 1% 1987 media, a small amount of growth was

observed in the 5% flask and good growth was observed in the 10% and 25% flasks.

Because 1987 media is an undefined media, the smallest amount possible was desired and

it was decided to use a 10% concentration when growing L. discophora in broths.

4.2 Bioreactor Design and Construction

While bacterial growth experiments were taking place, the bioreactor apparatus

for biofilm growth and adsorption experiments was designed and constructed. Two

complete setups were constructed so that a control could be run at the same time as the

actual experiment. All components needed to be autoclavable so that the entire

bioreactor apparatus could be sterilized before each experiment. The design included a

reservoir of inoculated media where temperature and pH could be monitored and

adjusted. Inoculated media was then pumped to the top of each column with a packed-

bed that the biofilm could be grown on. The media then returned to the reservoir in a

closed loop.

Two 2-L jacketed beakers with rubber lids were used as the media reservoirs

(Figure 2). Small holes of different sizes in the top of the rubber lids allowed for things

to go in and out of the reservoirs. A long glass thermometer was placed into each

reservoir to measure temperature, and an autoclavable pH probe was placed into each

reservoir to measure pH. A quarter-inch glass tube about four inches long with a rubber

lid was also placed into each lid. This allowed for addition of nutrients to encourage

growth and acids and bases to adjust pH to the reservoirs. The glass tube could also be

flamed after each time it was opened and something was added to help maintain sterile

conditions. A sampler was placed into each beaker capable of removing a small amount

24

of liquid from the reservoir without contamination. And finally, a long piece of quarter-

inch Tygon® tubing was run from the bottom of each reservoir through a peristaltic

pump and into the top of each column, and another long piece of Tygon® tubing was run

from the bottom of the column back into the reservoir. Everything was fit tightly into the

rubber lid and remaining holes were sealed off with rubber stoppers and solid glass rods

so no contamination could get into the reservoirs. Temperature-controlled water was run

through the jacket of the beaker from bottom to top to keep a steady reservoir

temperature of 28oC. The entire beaker was set on a stir plate and a Teflon® stir rod was

placed in the beaker to ensure even mixing inside the reservoir.

Figure 2 Reservoir Design

Stir Plate

Jacketed Beaker

Water In

Water Out

pH Probe

Thermometer

Media In From Column

Media Out To Column

Sampler

Glass Tube

Rubber Lid

25

The design for the two packed-bed columns where the L. discophora biofilms

would be grown is shown in Figure 3. Each column needed a place for the inoculated

media to enter at the top and exit at the bottom, as well as a place for air to be pumped in

and out. Two plastic Drierite® columns were used for the columns in the bioreactor

apparatus. The columns were emptied and then two quarter-inch holes were drilled in

each column. One hole was drilled in the center of the lid to allow media to enter and

another hole was drilled in the side of the column about two inches from the bottom to

allow air to enter. Existing holes near the top and bottom of the columns would allow

media and air to exit. Drilled holes were then threaded so that plastic fittings could be

screwed into the columns. The quarter-inch Tygon® tubing from the peristaltic pump

was connected to the fitting in the lid of each column and the existing hole near the

bottom of the column was connected to the tubing that ran back into the reservoir. An

aquarium air pump with an in-line filter was connected to the fitting two inches from the

bottom of the column with eighth-inch Tygon® tubing and more of the same tubing with

another in-line filter was connected to the existing hole near the top of the column.

26

Figure 3 Packed-bed Biofilm Column Design

In order to keep the column bed off the bottom of the column and allow the media

to drain out easily, a column support was constructed by cutting a circle the same

diameter as the inside of the column out of plastic. Many holes were punched in the lid

to allow the media to flow through but still keep the bed supported. In order to distribute

the media evenly over the top of the bed a showerhead style sprayer was constructed out

of a media bottle cap. Small holes were drilled in the cap and it was glued upside down

to the bottom of the column lid.

Quarter-inch solid polypropylene balls were chosen to make up the bed. Round

balls were chosen to provide a large surface area for the biofilm to grow on. A plastic

bed was desired because it would not adsorb the trace metals being tested and

polypropylene was good because it was autoclavable. Approximately 2,500 balls were

Media In

Media Out

Air Out

Air In

Column

Column Lid

Column Bed

Column Support

Air Filter

Air Filter

Sprayer

27

used in each bioreactor bed for a total bed volume of 0.475 L and a pore volume of 0.140

L. Once finished, the columns were zip-tied to a peg board stand and connected to the

reservoirs. The finished bioreactor apparatus setup is shown in Figure 4.

Figure 4 Bioreactor Apparatus

Packed-bed Columns

Aquarium Pumps

pH Controllers

Peristaltic Pump

Pump Controller

Reservoirs

Stir Plates

28

4.3 Bioreactor Operation

Bioreactor operation included the growth of the L. discophora biofilms and the

oxidation of manganese. Before this could begin the entire bioreactor apparatus needed

to be sterilized, pH probes needed to be calibrated, and the growth media and inoculation

broth for the bioreactors needed to be prepared. Two 100-mL broths of pure L.

discophora were grown from the same source in two 250-mL Erlenmeyer flasks on a

shaker table at 100 rpm over three days. MMS growth media with 10% 1987 media was

used to prepare the inoculation broths. At this point the broths were visibly turbid and

microscopic slides were prepared to make sure they were pure L. discophora. Two liters

of growth media were also prepared for the bioreactor reservoirs. MMS growth media

with 10% 1987 media was also used for this.

Before being sterilized, the pH probes were calibrated using standard buffer

solutions of pH 7 and pH 10. The probes were set to pH 7 and the slope was adjusted to

pH 10 in order to calibrate. To sterilize the bioreactor apparatus, the columns were

removed from the peg board, air pumps were unhooked at the filters, the tubing was

taken out of the peristaltic pump, and the entire setup was autoclaved at 121 oC for 15

minutes. The lids of the columns were loosened before autoclaving and retightened

immediately afterward to keep them from sealing shut. One of the inoculation broths was

poured into each liter of fresh media and then one liter of inoculated media was poured

into each reservoir of the bioreactor apparatus. This was all done under a laminar flow

hood to avoid contamination of any kind. The rubber lids of the reservoirs were then

taped shut with autoclave tape to provide a seal against contamination getting into the

reactors during operation. The columns were zip tied back up to the peg board, the

29

reservoirs were placed back on the stir plates, air pumps were hooked back up to the

filters, and the tubing was run back through the peristaltic pump.

4.3.1 Biofilm Growth

After sterilizing and setting up as described above, the bioreactors were ready to

begin growing L. discophora biofilms. The pH probes were hooked up, and the stir

plates were turned on and adjusted to 300 rpm to keep the reservoirs well mixed. The air

pumps to the columns were turned on to aerate the column beds. The temperature

controller was turned on, and water was pumped through the jacketed beakers in series

from bottom to top at a temperature of 28 oC. Finally, the peristaltic pump was turned on

and adjusted to setting 3 to bring the inoculated media from the reservoirs to the top of

the columns. A setting of 3 on the peristaltic pump corresponded to a flow rate of

approximately 50 mL per minute. The bioreactor apparatus was then checked for leaks

and if any were found the appropriate fittings were tightened.

Samples were taken from each reservoir as follows: the valve on the sampler was

opened, the sampler ball was squeezed once and released, filling a sterilized vial with

media, the valve was closed, the vial was removed and capped, and a new sterilized vial

was screwed onto the sampler. The pH probe was re-calibrated by collecting a sample

and testing for pH with a separate external pH probe and adjusting the set knob on the pH

meters accordingly. The pH of the media in the bioreactor reservoirs was then adjusted.

A solution of 1% nitric acid was made by adding 1 mL of nitric acid to 99 mL of DI

water, and a solution of 1% sodium hydroxide was made by adding 1 g of sodium

hydroxide to 99 mL of DI water. The rubber cap was taken off of the glass tube in the

reservoir lid and 1% nitric acid or 1% sodium hydroxide was added 1 mL at a time by

30

pipette until the pH in each bioreactor reservoir read 7.0. The top of the glass tube was

then flamed and the rubber cap was replaced. The pH of the bioreactor reservoirs was

adjusted in this way nearly every day. The pH tended to rise as the L. discophora grew,

but the pH was kept at 7 by manual adjustment.

Samples of the reservoir media were taken every four or five days to check the

accuracy of the pH probes and the health and purity of the bacteria. Microscopic slides

were prepared from the samples and viewed under the microscope at 1000X

magnification and photomicrographs were taken of the bacteria.

After six or seven days of biofilm growth an added shot of pyruvate was

introduced to each bioreactor reservoir in order to get fresh nutrients to the biofilm and

encourage growth. A 15 g/L solution of pyruvate was filter sterilized into a sterile bottle

and stored in the refrigerator. 150 mg of pyruvate was then added to each bioreactor

reservoir by adding 10 mL of the 15 g/L pyruvate solution. This was added by pipette

through the glass tube in the reservoir lid in the same way that pH was adjusted. This

process was repeated after another six or seven days to continue growth of the biofilms

until the end of the experiment.

The bioreactors were operated in this way for approximately three weeks. After

that it was decided that they were ready to begin manganese oxidation

4.3.2 Manganese Oxidation

A stock solution of 8.45 g/L MnSO4 · H2O was prepared by adding 0.845 g of

MnSO4 · H2O to 100 mL of DI water and filter sterilizing. 1 mL of this solution was then

added to one of the bioreactor reservoirs. This made a manganese concentration in the

reservoir of 50 uM. The second bioreactor reservoir was left without manganese to act as

31

a control. The difference between the two bioreactor setups was that one contained

biogenic manganese oxides in the biofilm of the column bed and the other one did not.

The biofilms were then given one day’s time to oxidize the manganese before conducting

adsorption experiments. Because of low adsorption to the manganese oxide biofilms in

the first experiment the concentration of manganese added to the bioreactor reservoir was

changed to 500 uM for all subsequent experiments. To do this, 10 mL of 8.45 g/L

MnSO4 · H2O was added to one of the bioreactor reservoirs instead of 1 mL. After the

biofilms had oxidized the manganese as much as possible toxic trace metal adsorption

was then ready to begin.

4.4 Lead Adsorption

To get the columns ready for lead adsorption experiments, the peristaltic pump,

air pumps, temperature control, pH probes and stir plates were all turned off and the

bioreactor reservoirs were disconnected from the columns. The air pumps and filters

were also disconnected from the columns and the loose ends of tubing were crimped off.

A 1-L solution of MMS with no phosphate, pyruvate, or vitamin B12 added was prepared

and pumped through the column bed in the up-flow direction (Figure 5). The MMS

solution was used to flush the bioreactor in preparation for lead adsorption experiments.

The up-flow allowed the bioreactor bed to be completely submerged in the MMS

solution. The solution was pumped at a rate of 5 mL/min for one hour. This flow rate

gave a retention time in the bioreactor bed of 30 minutes and ensured the column beds

were completely flushed after one hour.

To prepare the lead solution, several more liters of the MMS solution with no

phosphate, pyruvate, or vitamin B12 were prepared. A 2 uM or 414 ppb lead solution was

32

then prepared by adding 414 uL of 1000 ppm lead stock solution to each liter of MMS

solution. The lead solution was then pumped upwards through the columns at a rate of 5

mL/min. 10-mL samples of the effluent flow coming out the top of the columns were

taken in small polypropylene scintillation vials every 30 minutes until 25 samples were

taken from each column.

Figure 5 Up-Flow Lead Adsorption Column

4.5 Atomic Absorption Spectroscopy

Atomic absorption spectroscopy was used to determine the lead concentrations in

the samples taken from the columns as well as the manganese concentration on the

column bed after experimentation was complete. A Perkin-Elmer 3110 Atomic

Absorption Spectrometer with a Perkin-Elmer HGA-600 power source and a Perkin-

Lead Solution

Out

Lead Solution

In

Samples

Column Bed w/ Biofilm

33

Elmer AS-60 Autosampler were used for this analysis. Atomic absorption was chosen as

the method for analysis in this experiment because of its ability to measure extremely

small trace metal concentrations, single parts per billion, by graphite furnace atomic

absorption spectroscopy (GFAAS) as well as higher concentrations in parts per million,

by flame atomic absorption spectroscopy (FAAS). This versatility made atomic

absorption ideal for these analyses.

4.5.1 Lead Analysis by Atomic Absorption

To measure the lead concentrations in the effluent samples collected from the

column, GFAAS was used. Standard solutions of 0, 5, 10, 20, 50, 100 and 200 ppb lead

were prepared by adding different volumes of a 1000 ppb lead solution to MMS with no

phosphate, pyruvate, or vitamin B12. 2 mL of 1000 ppb lead solution were added to 8 mL

of MMS to make the 200 ppb standard, 1 mL of 1000 ppb lead solution was added to 9

mL of MMS to make the 100 ppb standard, and so on until only 10 mL of MMS was used

to make the 0 ppb standard. The standards were then analyzed by GFAAS to develop a

standard curve.

Before GFAAS analysis could be performed several steps needed to be taken to

prepare the machine. The graphite furnace autosampler unit was positioned into the

atomic absorption spectrometer and locked into place. The autosampler feed bottle was

filled with a 2% nitric acid in DI water solution and the liquid in the waste bottle was

properly disposed of. Argon, the venting fan and cooling water to the furnace from a tap

were turned on. The computer, spectrometer and power source were also turned on, and

the analysis software was loaded. The absorbance wavelength on the spectrometer was

set to 283.3 nm and the slit width was set to 0.7 inches. The lamp used for lead analysis

34

was then plugged in and the lamp adjustment tool on the software was selected. The

lamp was turned on and adjusted for maximum power. The furnace tool on the software

was then selected, the sampler tip was aligned into the graphite tube and the tube was

conditioned. At this point the spectrometer was ready to begin analyzing lead samples.

Using a pipette, 1 mL of each lead standard solution was put into a sample vial

and placed in the sampler. The method editor tool on the software was selected and lead

analysis was selected. This loaded the method and specifications for lead analysis. A

complete list of the conditions for graphite furnace atomic absorption analysis of lead is

included in Figure 6. The auto tool was then selected, locations for sampling were input,

and the analysis of the lead standards was begun. A small mirror was used to look into

the graphite tube to make sure that the sample was properly injected. The results tool on

the software was then selected and the absorbance of each standard was recorded. A

graph of absorbance vs. lead concentration was made from the data and an equation was

derived from the best fit line. The equation related lead concentration to absorbance and

could then be used to find the lead concentration of unknown samples.

The samples collected of the adsorption column effluent were analyzed by the

same method as the lead standards. The absorbance results were converted into ppb

using the standard curve equation and a graph of ppb lead coming out of the column over

time was generated.

35

Wavelength (nm): 283.3 Low Slit (nm): 0.7

Pretreatment Temp. (oC): 850 Atomization Temp. (oC): 1800

Tube/Site: Pyro/Platform Matrix Modifier: 0.2 mg NH4H2PO4

Characteristic Mass: 10.0 pg/0.0044 A-s Sensitivity Check: 22.7 ug/L for 0.2 A-s

Figure 6 Lead Graphite Furnace Atomic Absorption Conditions

4.5.2 Manganese Analysis by Atomic Absorption

After lead adsorption was complete, further analysis was conducted on the

biofilms in the column to determine the amount of manganese that was oxidized and

contained in the biofilms. After the column bed was dried and weighed (discussed in the

next section), it was soaked in 1 liter of a 2% nitric acid DI water solution. The solution

and column bed were poured into a 2 liter media bottle and periodically shaken over

several days to completely dissolve all the oxidized manganese in the biofilm. This

solution was then analyzed by FAAS for manganese concentration. Standard solutions of

0, 1, 2, 5, and 10 ppm manganese were prepared by adding different volumes of a 100

ppm manganese stock solution that had previously been prepared to DI water. 1 mL of

100 ppm manganese solution was added to 9 mL of DI water to make the 10 ppm

standard, 0.5 mL of 100 ppm manganese solution was added to 9.5 mL of DI water to

make the 5 ppm standard, and so on until only 10 mL of DI water was used to make the 0

ppm standard. The standards were then analyzed by FAAS to develop a standard curve.

Before FAAS analysis could be performed several steps needed to be taken to

prepare the instrument. The graphite furnace autosampler unit was removed from the

atomic absorption spectrometer and replaced with the flame unit. Connections for the

36

fuel, oxidant, interlocks and interface were made before the flame unit was positioned

and screwed into place. The lamp used for lead analysis was removed and replaced with

the lamp used for manganese analysis. Operation of the atomic absorption spectrometer

for FAAS was performed from the interface on the spectrometer rather than from the

computer. The lamp was adjusted to maximize power, the absorbance wavelength was

set to 278.9 nm and the slit width was set to 0.2 inches. At this point the spectrometer

was ready to begin analyzing manganese samples.

The atomic absorption spectrometer was turned on and acetylene and oxygen

flows were initiated. The venting fan was turned on before the lighting of the flame. To

light the flame, the control knob on the spectrometer was turned to the setting for air as

an oxidant. The fuel level was adjusted to a setting of 3, and the oxidant level was

adjusted to a setting of 5. The ignite button was pressed and held until the flame was lit.

The sampling tube was then dipped into each of the manganese standards and the

absorption was read directly off the spectrometer interface. A standard curve of

absorbance vs. manganese concentration was made from the data and an equation was

derived from the best fit line.

With a standard curve for manganese concentration complete, the analysis of the

solution from the column bed with unknown manganese concentration could then be

completed. The sampling tube was dipped into the solutions and the absorbance was

recorded. These data were then converted into ppm manganese using the standard curve

equation.

Because the manganese concentrations of the samples were not within the range

of the standard curve, GFAAS was then performed on the solution. Manganese standards

37

of 0, 5, 20, 50, 200 and 500 ppb were prepared in the same way the other previous

manganese standards had been prepared. The flame unit was replaced with the graphite

furnace autosampler unit and set up in the same way as before only the manganese lamp

was left in and the corresponding wavelength remained the same. The standards were

analyzed in the same way as the lead standards except that the manganese method was

loaded on the software instead of the lead method. A complete list of the conditions for

graphite furnace atomic absorption analysis of manganese is included in Figure 7. The

standard curve and best fit equation were also generated in the same way. The solution

from the column bed with unknown manganese concentration was then tested by GFAAS

and the result was recorded. The graphite furnace was then used in all subsequent tests

for manganese concentration.

Wavelength (nm): 279.5

Low Slit (nm): 0.2 Pretreatment Temp. (oC): 1400 Atomization Temp. (oC): 2200

Tube/Site: Pyro/Platform Matrix Modifier: 0.5 mg Mg(NO3)2

Characteristic Mass: 2.0 pg/0.0044 A-s Sensitivity Check: 4.5 ug/L for 0.2 A-s

Figure 7 Manganese Graphite Furnace Atomic Absorption Conditions

4.6 Biomass Characterization

To characterize the biofilm in the column bed, the concentration of bacteria in the

biofilm needed to be found in addition to the oxidized manganese concentration in the

biofilm. With this information the biomass could be completely characterized in terms of

the amount of bacteria grown, the amount of manganese oxidized, and the amount of lead

adsorbed. Two methods were used in an attempt to find the concentration of bacteria in

38

the biomass. First, microscopy was used to count the number of cells in the biofilm and

second, dry weight was used to find the mass of the biofilm.

4.6.1 Microscopy

At the completion of lead adsorption, a single bead was taken out of the column

bed. The bead was placed on a microscopic slide and a cover slip was balanced on top of

the bead. The slide was placed under the microscope and viewed at 1000X

magnification. Attempts were made to count the number of bacteria on the surface of the

bead over a certain surface area, but this turned out to be most difficult due to clumping

of the bacteria and non-uniform growth over the bead surface. This method of

characterizing the concentration of bacteria in the biomass was quickly abandoned and

the method of dry weight analysis was used instead.

4.6.2 Dry Weight

After lead adsorption was complete, the air pumps were reconnected to the

columns and turned on. Air was pumped through the columns for several days until the

column beds had completely dried out. The column bed was then removed from the

column and weighed before being immersed in 2% nitric acid for manganese testing.

After the manganese testing was completed, the beads were thoroughly washed and

completely dried again in the same way as before. The beads were then weighed a

second time and the difference in weights represented biofilm weight. In subsequent

experiments the bioreactor bed was divided into two halves and weighed separately in an

attempt to provide more accurate results. The two halves were also tested separately for

manganese concentration by GFAAS.

39

The entire process of growing biofilms, oxidizing manganese, adsorbing lead, and

analyzing the samples and column bed was repeated three times from beginning to end.

Slight changes, as noted in the methods, were made after each complete run in an attempt

to maximize lead adsorption to the column bed. In the third run, manganese was added to

both bioreactors and no control was run. The results of all three runs are detailed in the

next chapter.

40

CHAPTER 5

RESULTS

The results chapter is divided into three main sections, one for each of the runs

completed in the project. Data for the runs are referred to by the dates that lead

adsorption and analysis were performed. The first section shows the results from the first

run in which analysis was performed on 8/17/2004, the second section shows results from

the second run in which analysis was performed on 11/6/2004, and the third section

shows the results from the third run in which analysis was performed on 12/3/2004. Each

of these three sections is divided into subsections which show the results from analysis of

lead atomic absorption, manganese atomic adsorption, biomass dry weight, and

observations and microscopy. A fourth section at the end of the chapter outlines results

for the overall success of the biofilm reactor by comparing results from all three runs.

5.1 Results from First Complete Run

The first complete run was really the third attempt to grow and test a biofilm

using the bioreactor apparatus. The first attempt was aborted because the peristaltic

pump wore through the tubing after about a week and all the media was pumped out all

over the lab space. The second attempt was aborted because of high levels of microbial

contamination and low levels of L. discophora growth in the media. Operation and

growth on the third attempt was much improved and this turned out to be the first

complete run. Growth of the biofilm on the column bed began by inoculation of the

media reservoir on 7/30/2004. 2.44 mg of manganese (44 umol/L) was added to one of

the media reservoirs after 18 days on 8/16/2004, and 24 hours was allowed for

41

manganese oxidation. On 8/17/2004 a lead solution was pumped through the columns,

and lead analysis was performed on the effluent samples the same day.

5.1.1 Lead Breakthrough Curve for First Run

A lead solution of concentration 414 ppb (2 umol/L) was run through both

columns at 5 mL/min and 10 mL samples were taken every 30 minutes. The total liquid

volume in the packed-bed column was 140 mL, so 30 minutes equals about 1 retention

time. These samples were then tested for lead concentration by GFAAS. The atomic

absorption results of lead standard analysis are shown in Table 3 and Figure 8. A linear

regression of the standard data gave the equation shown in Figure 8 with an R2 of 0.9995.

Table 3 Results from Lead Standards by GFAAS from 8/17/2004

LEAD STANDARDS 8/17/2004 ppb Pb Abs #1 Abs #2 Abs#3 Avg Abs Std. Dev.

0 0.010 0.006 0.003 0.006 0.004 5 0.016 0.015 0.017 0.016 0.001

10 0.030 0.024 0.017 0.024 0.007 20 0.049 0.046 0.040 0.045 0.005 50 0.105 0.088 0.088 0.094 0.010 100 0.189 0.167 0.176 0.177 0.011 200 0.339 0.330 0.339 0.336 0.005 414 0.558 0.538 0.534 0.543 0.013

42

y = 607x - 5.5268R2 = 0.9995

0

50

100

150

200

250

0.0 0.1 0.2 0.3 0.4 0.5

Absorbance

Pb c

once

ntra

tion

(ppb

)

Figure 8 Lead Calibration Curve by GFAAS from 8/17/2004

The standard curve was used to calculate the lead concentration of each effluent

sample collected, and these results for column #1, the column where manganese was

added to the media, are shown in Table 4. The results for the same test from column #2,

the control column with no manganese added, are shown in Table 5.

43

Table 4 Results for Lead Samples from Column #1, Manganese Added, by GFAAS from 8/17/2004

SAMPLE 8/17/2004

time (hrs) Abs #1 Abs #2 Avg Abs ppb Pb 0.0 0.001 0.000 0.001 -5 0.5 -0.008 -0.011 -0.010 -11 1.0 -0.017 -0.005 -0.011 -12 1.5 0.042 0.017 0.030 12 2.0 0.071 -0.007 0.032 14 2.5 0.112 0.044 0.078 42 3.0 0.188 0.099 0.144 82 3.5 0.234 0.139 0.187 108 4.0 0.277 0.202 0.240 140 4.5 0.279 0.191 0.235 137 5.0 0.259 0.284 0.272 159 5.5 0.337 0.329 0.333 197 6.0 0.312 0.329 0.321 189 6.5 0.330 0.308 0.319 188 7.0 0.325 0.338 0.332 196 7.5 0.348 0.353 0.351 207 8.0 0.367 0.355 0.361 214 8.5 0.388 0.377 0.383 227 9.0 0.388 0.428 0.408 242 9.5 0.423 0.424 0.424 252 10.0 0.518 0.538 0.528 315 10.5 0.523 0.531 0.527 314 11.0 0.536 0.570 0.553 330 11.5 0.527 0.560 0.544 324 12.0 0.557 0.576 0.567 338

44

Table 5 Results for Lead Samples from Column #2, Control with No Manganese Added, by GFAAS from 8/17/2004

CONTROL 8/17/2004

time (hrs) Abs #1 Abs #2 Avg Abs ppb Pb 0.0 0.000 -0.002 -0.001 -6 0.5 0.003 -0.001 0.001 -5 1.0 -0.008 -0.008 -0.008 -10 1.5 0.089 0.006 0.048 23 2.0 0.120 0.097 0.109 60 2.5 0.195 0.083 0.139 79 3.0 0.293 0.176 0.235 137 3.5 0.369 0.207 0.288 169 4.0 0.380 0.261 0.321 189 4.5 0.379 0.350 0.365 216 5.0 0.457 0.376 0.417 247 5.5 0.498 0.444 0.471 280 6.0 0.465 0.437 0.451 268 6.5 0.466 0.468 0.467 278 7.0 0.468 0.496 0.482 287 7.5 0.317 0.164 0.241 140 8.0 0.539 0.469 0.504 300 8.5 0.563 0.439 0.501 299 9.0 0.577 0.518 0.548 327 9.5 0.605 0.512 0.559 333 10.0 0.670 0.618 0.644 385 10.5 0.663 0.505 0.584 349 11.0 0.656 0.575 0.616 368 11.5 0.653 0.533 0.593 354 12.0 0.640 0.479 0.560 334

The results of the samples from both columns were then graphed to show the

breakthrough curves for lead by the column beds over time and the difference between

the control column bed and the sample column bed containing oxidized manganese

(Figure 9). The graph shows that lead was adsorbed for 2 hours time before

concentrations began to steadily increase and level off after 10 hours at about 350 ppb.

45

-50

0

50

100

150

200

250

300

350

400

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Time (hrs)

Pb c

once

ntra

tion

(ppb

)

L. discophoraonly (Control)L. discophorawith Mn oxide

Figure 9 Breakthrough Curves for L. discophora Lead Adsorption Over Time by Both Control and Sample Column Packed-beds With and Without Manganese Oxide from 8/17/2004

5.1.2 Manganese Analysis for First Run

The manganese content of the biofilms was determined by extracting the

manganese into nitric acid at the end of the experiment and measuring manganese

concentration by GFAAS. Manganese standards were prepared and analyzed using

GFAAS (Table 6), and these results were then graphed to create a calibration curve for

manganese over a 5 to 500 ppb range (Figure 10).

46

Table 6 Results from Manganese Standards by GFAAS from 8/17/2004

Mn STANDARDS 8/17/2004 ppb Mn Abs #1 Abs #2 Avg Abs Std. Dev.

5 0.036 0.081 0.059 0.032 20 0.147 0.218 0.183 0.050 50 0.430 0.467 0.449 0.026

200 1.186 1.077 1.132 0.077 500 1.339 1.465 1.402 0.089

Sample 0.899 1.040 0.970 0.100

y = 322.92x - 53.155R2 = 0.8486

-100

0

100

200

300

400

500

600

0.000 0.500 1.000 1.500

Absorbance

Mn

conc

entra

tion

(ppb

)

Figure 10 Manganese Calibration Curve by GFAAS from 8/17/2004

47

Also shown with the manganese standards in Table 6 is the absorbance of the

manganese sample taken from the column bed. This absorbance was then converted to a

concentration using the equation in Figure 10 resulting in a manganese concentration of

260 ppb when dissolved in 1 L of 2% nitric acid. This concentration corresponds to 0.26

mg of oxidized manganese on the column bed and a surface concentration of 0.82 mg/m2

or 15 umol/m2. Since 2.44 mg total manganese was added to the broth, only about 10%

of the manganese ended up in the biofilms.

5.1.3 Biomass Dry Weight for First Run

To determine the weight of the biomass on the column bed, a dry weight method

of analysis was used. The total dry weight of biofilm in the column was 0.22 g (Table 7)

and the concentration of manganese in the biofilm was 1.18 mg Mn/g biofilm.

Table 7 Biomass Dry Weight from 8/17/2004

BIOMASS 8/17/2004 Dry Wt. w/ Biofilm (g) 270.60 Dry Wt. w/o Biofilm (g) 270.38 Biomass Wt. (g) 0.22

48

5.1.4 Observations and Microscopy for First Run

During the course of biofilm growth both visual observations of the broth in the

reservoir and microscopic observations of samples of that broth were made. In the

sample reactor from 8/17/2004 it was noted that growth appeared to be pure L.

discophora. This can be seen in photomicrographs taken of the broth at magnification

1000X (Figure 11).

Figure 11 1000X Magnification of Media Sample Taken from the Sample Column on 8/17/2004

49

In the control reactor from 8/17/2004 it was noted that growth appeared to be

predominately L. discophora, but that some contamination was present. This can be seen

in photomicrographs taken of the broth at magnification 1000X (Figure 12).

Figure 12 1000X Magnification of Media Sample Taken from the Control Column on 8/17/2004

50

5.2 Results from Second Complete Run

The second complete run also took a couple of attempts to get started before an

entire run was completed. The first attempt was aborted because of severe microbial

contamination in both the control and sample bioreactors. Growth on the second attempt

was much improved and this became the second complete run. Growth of the biofilm on

the column bed began by inoculation of the media reservoir on 10/18/2004. 27.5 mg of

manganese (500 umol/L) was added to one of the media reservoirs after 18 days on

11/5/2004, and 24 hours was allowed for manganese oxidation. On 11/6/2004 a lead

solution was pumped through the columns, and lead analysis was performed on the

effluent samples the same day. Increased levels of manganese were used in order to get

the maximum oxidized manganese concentration possible on the column bed and

therefore the maximum lead adsorption.

5.2.1 Lead Breakthrough Curve for Second Run

A lead solution of concentration 414 ppb (2 umol/L) was again run through both

columns at 5 mL/min and 10 mL samples were taken every 30 minutes. The effluent

samples were then tested for lead concentration by GFAAS. New lead standards were

also prepared and analyzed by GFAAS. The atomic absorption results of lead standard

analysis are shown in Table 8 and Figure 13. A linear regression of the standard data

gave the equation shown in Figure 13 with an R2 of 0.9954.

51

Table 8 Results from Lead Standards by GFAAS from 11/6/2004

LEAD STANDARDS 11/6/2004 ppb Pb Abs #1 Abs #2 Avg Abs Std. Dev.

0 0.001 -0.012 -0.006 0.009 5 0.011 0.021 0.016 0.007

10 0.018 0.034 0.026 0.011 20 0.064 0.049 0.057 0.011 50 0.128 0.131 0.130 0.002 100 0.272 0.270 0.271 0.001 200 0.463 0.491 0.477 0.020

y = 411.37x - 2.034R2 = 0.9954

0

50

100

150

200

250

0.0 0.1 0.2 0.3 0.4 0.5

Absorbance

Pb c

once

ntra

tion

(ppb

)

Figure 13 Lead Calibration Curve by GFAAS from 11/6/2004

The breakthrough curve results for column #1, the column where manganese was added

to the media and column #2, the control column with no manganese added, are shown in

Table 9.

52

Table 9 Effluent Lead Concentrations from Column #1, Manganese Added, and Column #2, Control with No Manganese Added, by GFAAS from 11/6/2004

The effluent lead concentrations from both columns were then graphed to show the

adsorbance of lead by the column beds over time and the difference between the control

column bed and the sample column bed containing oxidized manganese (Figure 14).

Results showed that immediately the first effluent samples collected after 30 minutes had

lead concentrations of about 300 ppb and these remained constant throughout the 12-hour

experiment. The first samples taken from each column showed lower concentrations of

CONTROL 11/6/2004 SAMPLE 11/6/2004 time (hrs) Abs ppb Pb time (hrs) Abs ppb Pb

0.0 0.232 93 0.0 0.075 29 0.5 0.688 281 0.5 0.612 250 1.0 0.753 308 1.0 0.708 289 1.5 0.754 308 1.5 0.698 285 2.0 0.742 303 2.0 0.697 285 2.5 0.735 301 2.5 0.701 286 3.0 0.736 301 3.0 0.714 292 3.5 0.716 293 3.5 0.729 298 4.0 0.690 282 4.0 0.672 274 4.5 0.691 282 4.5 0.667 272 5.0 0.681 278 5.0 0.708 289 5.5 0.764 312 5.5 0.760 311 6.0 0.691 282 6.0 0.795 325 6.5 0.765 313 6.5 0.773 316 7.0 0.735 301 7.0 0.654 267 7.5 0.768 314 7.5 0.695 284 8.0 0.698 285 8.0 0.720 294 8.5 0.722 295 8.5 0.699 286 9.0 0.743 304 9.0 0.766 313 9.5 0.920 377 9.5 0.781 319

10.0 0.759 310 10.0 0.795 325 10.5 0.758 310 10.5 0.775 317 11.0 0.748 306 11.0 0.772 316 11.5 0.742 303 11.5 0.668 273 12.0 0.757 310 12.0 0.771 315

53

lead only because columns were initially full of MMS media and the lead solution hadn’t

completely filled the column yet.

-50

0

50

100

150

200

250

300

350

400

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Time (hrs)

Pb c

once

ntra

tion

(ppb

)

L. discophora only(Control)L. discophora with Mnoxide

Figure 14 Breakthrough Curves for L. discophora Lead Adsorption Over Time by Both Control and Sample Column Packed-beds With and Without Manganese Oxide from 11/6/2004

5.2.2 Manganese Analysis for Second Run

The manganese content of the biofilms was again determined by extracting the

manganese with nitric acid at the end of the experiment and measuring manganese

concentration by GFAAS. New manganese standards were also prepared and analyzed

using GFAAS (Table 10), and these results were then graphed to create a new calibration

curve for manganese over a 0 to 500 ppb range (Figure 15).

54

Table 10 Results from Manganese Standards by Graphite Furnace Atomic Absorption Spectroscopy from 11/6/2004

Mn STANDARDS 11/6/2004

ppb Mn Abs #1 Abs #2 Avg Abs Std. Dev. 0 -0.213 0.173 -0.020 0.273 5 -0.176 0.006 -0.085 0.129 20 0.063 0.108 0.086 0.032 50 0.190 -0.022 0.084 0.150

200 0.530 0.401 0.466 0.091 500 0.892 0.588 0.740 0.215

Sample -0.380 0.517 0.069 0.634

y = 588.53x + 4.5946R2 = 0.9295

-100

0

100

200

300

400

500

600

-0.200 0.000 0.200 0.400 0.600 0.800

Absorbance

Mn

conc

entra

tion

(ppb

)

Figure 15 Manganese Calibration Curve by GFAAS from 11/6/2004

Many of the results in Table 10 have a very high standard deviation due to low

reproducibility during atomic absorption. This should be noted when considering the

results for manganese concentration. Also shown with the manganese standards in Table

10 is the absorbance of the manganese sample taken from the column bed. This

55

absorbance was then converted to a concentration using the equation in Figure 15

resulting in a manganese concentration of 45 ppb when dissolved in 1 L of 2% nitric acid.

This concentration corresponds to 0.045 mg of oxidized manganese on the column bed

and a surface concentration of 0.14 mg/m2 or 2.6 umol/m2, but as mentioned before these

values may not be very accurate due to high deviations between replicates for both the

manganese standards and the samples.

5.2.3 Biomass Dry Weight for Second Run

To determine the weight of the biomass on the column bed, a dry weight method

of analysis was again used. The weight of the column bed with the biomass on it was

taken after having been thoroughly dried, the biomass was then cleaned off of the column

bed, and the column bed was then reweighed. The weight before and after removing

biofilm was the same (Table 11), indicating that no measurable biomass had accumulated

on the beads during this run.

Table 11 Biomass Dry Weight from 11/6/2004

BIOMASS 11/6/2004 Dry Wt. w/ Biofilm 117.63gDry Wt. w/o Biofilm 117.63gBiomass Wt. 0.00g

56

5.2.4 Observations and Microscopy for Second Run

During the course of biofilm growth, both visual observations of the broth in the

reservoir and microscopic observations of samples of that broth were again made. In the

sample reactor broth from 11/6/2004 it was noted that growth appeared to be some L.

discophora with a lot of microbial contamination. This can be seen in photomicrographs

taken of the sample broth at magnification 1000X (Figure 16).

Figure 16 1000X Magnification of Media Sample Taken from the Sample Column from 11/6/2004

57

In the control reactor from 11/6/2004 it was noted that growth appeared to also be some

L. discophora with a lot of microbial contamination present. This can be seen in

photomicrographs taken of the control broth at magnification 1000X (Figure 17).

Figure 17 1000X Magnification of Media Sample Taken from the Control Column from 11/6/2004

Even though the measured weight of the biomass on the columns was zero, bacterial cells

were still present in the broth as can be seen in Figure 16 and Figure 17.

58

5.3 Results from Third Complete Run

A different method for bioreactor inoculation was used for the third complete run

to limit the amount of microbial contamination. Two full liters of L. discophora broth

were grown and poured into the reactors instead of inoculating sterile media from 100

mL broths. Growth of the biofilm on the column bed then began on 11/15/2004.

Another difference in the third compete run was that no control was used since sufficient

control data had been collected from previous experiments. 27.5 mg of manganese (500

umol/L) was added to both of the media reservoirs after 17 days on 12/2/2004, and 24

hours was allowed for manganese oxidation. On 12/3/2004 a lead solution was pumped

through the columns, and lead analysis was performed on the effluent samples the same

day.

5.3.1 Lead Breakthrough Curve for Third Run

A lead solution of concentration 414 ppb (2 umol/L) was again run through both

columns at 5 mL/min and 10 mL samples were taken every 30 minutes. The effluent

samples were then tested for lead concentration by GFAAS. New lead standard were

also prepared and analyzed by GFAAS. The atomic absorption results of lead standard

analysis are shown in Table 12 and Figure 18. A linear regression of the standard data

gave the equation shown in Figure 18 with an R2 of 0.9946.

59

Table 12 Results from Lead Standards by GFAAS from 12/3/2004

LEAD STANDARDS 12/3/2004 ppb Pb Abs

0 0.008 5 0.016

10 0.034 20 0.053 50 0.142 100 0.254 200 0.448 414 0.749

y = 445.82x - 5.8229R2 = 0.9946

0

50

100

150

200

250

0 0.1 0.2 0.3 0.4 0.5

Absorbance

Pb c

once

ntra

tion

(ppb

)

Figure 18 Lead Calibration Curve by GFAAS from 12/3/2004

60

The results from column #1 and column #2, both with manganese added to the media, are

shown in Table 13. The effluent lead concentrations from both columns were then

graphed to show the adsorbance of lead by the column beds over time (Figure 19).

Results showed that some lead began coming through the column immediately and then

steadily increased until leveling off after about 4 hours.

Table 13 Results for Lead Samples from Column #1 and Column #2, Both with Manganese Added, by GFAAS from 12/3/2004

COLUMN #1 12/3/2004 COLUMN #2 12/3/2004

time (hrs) Abs ppb Pb time (hrs) Abs ppb Pb 0.0 -0.006 -8 0.0 0.016 1 0.5 0.003 -4 0.5 0.043 13 1.0 0.123 49 1.0 0.367 158 1.5 0.315 135 1.5 0.472 205 2.0 0.419 181 2.0 0.549 239 2.5 0.501 218 2.5 0.553 241 3.0 0.587 256 3.0 0.553 241 3.5 0.577 251 3.5 0.618 270 4.0 0.593 259 4.0 0.621 271 4.5 0.665 291 4.5 0.654 286 5.0 0.663 290 5.0 0.706 309 5.5 0.675 295 5.5 0.678 296 6.0 0.663 290 6.0 0.685 300 6.5 0.667 292 6.5 0.670 293 7.0 0.718 314 7.0 0.660 288 7.5 0.711 311 7.5 0.705 308 8.0 0.728 319 8.0 0.714 312 8.5 0.720 315 8.5 0.706 309 9.0 0.682 298 9.0 0.717 314 9.5 0.727 318 9.5 0.722 316

61

-50

0

50

100

150

200

250

300

350

400

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Time (hrs)

Pb c

once

ntra

tion

(ppb

)

Column #1Column #2

Figure 19 Lead Breakthrough Curves for L. discophora Lead Adsorption Over Time by Both Packed-bed Columns With Manganese Oxide from 12/3/2004

5.3.2 Manganese Analysis for Third Run

The manganese content of the biofilms was again determined by extracting the

manganese with nitric acid at the end of the experiment and measuring manganese

concentration by GFAAS. This analysis was performed at the same time as that from the

second complete run and therefore the same manganese standards (Table 10) and

calibration curve for manganese over a 0 to 500 ppb range (Figure 15) were used. Again,

many of the results in Table 14 had a very high standard deviation due to low

reproducibility during atomic absorption and this should be noted when considering the

results for manganese concentration in the column.

62

To determine the manganese content of the columns on the third compete run,

each column bed was divided in half and both halves were extracted into 500 mL of nitric

acid and analyzed using GFAAS. The results from this analysis are shown in Table 15.

Absorbances were converted to concentrations using the equation in Figure 20 resulting

in manganese concentrations of 408 ppb for column #1 and 338 ppb for column #2 when

dissolved in 1 L of 2% nitric acid. These concentrations correspond to 0.41 mg of

oxidized manganese on the column bed for column #1 and 0.34 mg for column #2.

Surface concentrations were calculated to be 1.29 mg/m2 or 23 umol/m2 for column #1

and 1.07 mg/m2 or 19 umol/m2 for column #2.

Table 14 Results of Manganese Concentration on the Column Beds from 12/3/2004

SAMPLES 12/3/2004

Reactor Abs #1 Abs #2 Avg. ppb Mn Std. Dev. Avg. ppb Mn 1.1 0.697 1.049 0.873 518 0.249 1.2 0.433 0.562 0.498 297 0.091

408

2.1 0.990 0.688 0.839 498 0.214 2.2 0.082 0.509 0.296 179 0.302

338

63

5.3.3 Biomass Dry Weight

To determine the weight of the biomass on the column bed, a dry weight method

of analysis was again used. However, the weight was calculated for each half of both

column beds in order to achieve more precise results (Table 16). The weights from the

halves of each column were then added together to get the ultimate biomass on each

column. The total dry weight of biofilm in column #1 was 0.071 g and in column #2 was

0.094 g. The concentration of manganese in the biofilm of column #1 was 5.77 mg Mn/g

biofilm and in the biofilm of column #2 was 3.62 mg Mn/g biofilm.

Table 15 Biomass Dry Weights from 12/3/2004

BIOMASS 12/3/2004 Column Wt. 1 Wt. 2 Wt. (g) Total Wt. (g)

1.1 137.129 137.121 0.008 1.2 112.352 112.289 0.063

0.071

2.1 128.655 128.602 0.053 2.2 123.340 123.299 0.041

0.094

64

5.3.4 Observations and Microscopy for Third Run

During the course of biofilm growth both visual observations of the broth in the

reservoir and microscopic observations of samples of that broth were again made. In

column #1 from 12/3/2004 growth appeared to be predominately L. discophora with

some microbial contamination. This can be seen in photomicrographs taken of the broth

at magnification 1000X (Figure 21).

Figure 20 1000X Magnification of Media Sample Taken from the Sample Column #1 from 12/3/2004

65

In column #2 from 12/3/2004 that growth also appeared to be predominately L.

discophora with some microbial contamination present. This can be seen in

photomicrographs taken of the broth at magnification 1000X (Figure 22).

Figure 21 1000X Magnification of Media Sample Taken from the Sample Column #2 from 12/3/2004

66

5.4 Lead Removal Efficiency for All 3 Runs

In order to see how successful the experiments were it is useful to see all the lead

adsorption results together. A total of six biofilms were grown in the three complete runs

and the lead adsorption data from these were put onto a single graph (Figure 23).

-50

0

50

100

150

200

250

300

350

400

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Time (hrs)

Pb c

once

ntra

tion

(ppb

)

Control 8/17/2004 Sample 8/17/2004 Control 11/6/2004Sample 11/6/2004 Sample #1 12/3/2004 Sample #2 12/3/2004

Figure 22 Lead Adsorption Results from All 3 Runs

From Figure 23 it is clear that there was essentially no lead adsorption for the two runs on

11/6/2004, but some adsorption was observed for the other 4 runs. The lack of lead

adsorption observed for the 11/6/2004 experiment may have been due to the very low

biomass content during that experiment, as discussed in the next chapter. For the 4

67

column runs that exhibited lead adsorption, there was a very short period of low effluent

lead concentrations. This is an important limitation of these columns for practical lead

removal from wastewater, and this too is discussed in the following chapter.

68

CHAPTER 6

DISCUSSION

6.1 Lead Adsorption Results Discussion As described above and illustrated in Figure 23, the observed lead adsorption

resulted in breakthrough cures that would not be practical for removal of lead from

wastewater. There are many possible explanations for the poor breakthrough curves and

we will investigate many of these in this chapter. There were also large differences in the

breakthrough curves for each of the three pairs of column runs. In this chapter we first

compare the results of each completed run (Section 6.1) and examine possible limitations

to get a better understanding of the results.

The most lead adsorption was observed for the first complete run on 8/17/2004.

In this run there is a clear difference between the sample results, with manganese oxide,

and the control results, without manganese oxide (Figure 9). This difference shows that

there was increased adsorption due to the presence of biogenic manganese oxide as this

was the only difference between the two columns. The observations during initial

biofilm growth showed that these columns had the best growth with the highest

percentage of L. discophora and the least amount of microbial contamination. This is the

most plausible reason why the first complete run showed better results than the next two.

Results from the biomass dry weight and manganese analysis showed a biomass of 0.22

g, the highest recorded, and 0.26 g of manganese deposited on the column bed. It should

also be noted that this was the first time the columns and column beds had been exposed

to lead solutions and there may have been adsorption there that was not present in the

69

subsequent experiments. Although the apparatus was thoroughly washed and rinsed with

DI water between each experiment, there may have been some level of adsorption to the

column and the column bed upon first exposure that was not washed away. In future

experiments the apparatus should be acid washed between each run. This adsorption

could not account for the difference between the sample with manganese oxide and the

control without manganese oxide since the data would be skewed the same amount in

each column. However, this could be a factor in accounting for the increased adsorption

in the 8/17/2004 run as compared to the next two completed runs.

The least lead adsorption was observed for the second complete run on 11/6/2004.

Both sample and control in this run showed almost no adsorption (Figure 14). In fact, the

results are very close to what would be expected if there was no biofilm present on the

column bed of any kind and therefore no oxidized manganese either. The observations

during growth showed that these columns indeed had no measurable growth and high

levels of microbial contamination. Results from the biomass dry weight and manganese

atomic absorption analysis on the column bed confirmed these observations. Biomass

dry weight analysis showed no measurable biomass, and the total manganese deposited

on the column bed was also very small, measured at 0.045 g and this value is suspect due

to high deviation in the standards. These results show that both the sample column and

the control column from 11/6/2004 can be seen as control experiments in which there was

little to no biomass of any kind and almost no oxidized manganese present.

Results from the third complete run on 12/3/2004 are slightly different than the

first two in that there was no control column, but instead two sample columns both with

manganese oxide. This was done to increase the chances of one of the columns having

70

more significant growth of pure L. discophora. Both columns exhibited approximately

the same growth and very similar lead adsorption results. Observations during growth

showed that both columns had significant biofilm growth predominately made up of L

discophora, but with some microbial contamination. This made these columns

comparable to the sample column from 8/17/2004, however, the results showed less

adsorption than that column (Figure 23). In addition to the reasons already discussed,

there could be several explanations for this. Microbial contamination in the columns

from 12/3/2004 could make a significant difference since there was minimal

contamination in the sample column from 8/17/2004. The inoculation media was also

prepared differently in the third complete run by growing the entire liter of reservoir

media in a media bottle on a shaker table and this could have changed biofilm growth and

manganese oxidation.

It is also useful to see what the bacteria in the media reservoirs, seen in

photomicrographs taken from each completed run, looked like compared to what pure

cultures of L. discophora from the inoculating broths and pure cultures of L. discophora

from plates with oxidized manganese looked like. There is a much higher density of

bacteria in these pictures and the bacterial cultures appear to be much healthier with no

microbial contamination present. Some differences in appearance can be attributed to the

different growth conditions in the bioreactor apparatus as opposed to the broths and

plates, but ideally the bacteria would look very similar. Photomicrographs taken at

magnification 1000X of pure L. discophora cultures from an inoculation broth, Figure 24,

and from a growth plate with oxidized manganese present, Figure 25, are shown below.

71

Figure 23 1000X Magnification of pure L. discophora from Inoculation Broth

Figure 24 1000X Magnification of pure L. discophora from Growth Plate with Oxidized Manganese Present

72

The total biomass and manganese in the columns from all completed runs is

compared in Table 17. The results for the first and third complete runs are comparable

while the second complete run showed little or no biomass or manganese. Increased

manganese oxide deposits compared to total biomass on the third run can be accounted

for because 10 times more manganese was added to those columns.

Table 16 Total Biomass and Manganese Results from All Complete Runs

Biomass (g) Mn Content (mg) 1st Run 0.22 0.26 2nd Run 0.00 0.05

3rd Run #1 0.07 0.41 3rd Run #2 0.09 0.34

Even understanding the differences between the lead adsorption results from the

three completed runs it is still important to explore methods of increasing lead adsorption

for more practical use of these columns. The first consideration is if the equilibrium

adsorption capacity of the biogenic manganese oxides is sufficient for the lead removal

desired. This is explored below in Section 6.2 by comparing the lead adsorption in these

experiments to that reported in the literature for biogenic manganese oxides. This will

tell us if enough manganese oxide was provided on the biofilms. The second

consideration is the kinetics of lead adsorption in the columns. It is possible that kinetic

and mass-transfer limitations do no allow enough time for the desired lead adsorption. If

the kinetics of lead adsorption are slow, it is possible that it is just not feasible given the

apparatus and conditions used in this experiment to provide useful breakthrough curves.

This is discussed in Section 6.3.

73

6.2 Comparison to Lead Adsorption by Manganese Oxides in Other Studies

Here lead adsorption observed in the columns in this research is compared to

other research that has been done on lead adsorption by biogenic manganese oxides. By

comparing the results from this research to similar research that has been done on the

subject it is possible to determine, at least part of the reason, why limited lead adsorption

was observed. In the following sections, the quantity of manganese oxide present in the

biofilms (Section 6.2.1) and the amount of lead adsorbed to that manganese oxide

(Section 6.2.2) are compared to results from previous research.

6.2.1 Quantity of Manganese in the Biofilms

Oxidized manganese concentrations from this research were compared to those

from other research performed to determine if there were much higher levels of

biogenically oxidized manganese in those experiments that were allowing an increased

level of lead adsorption. Previous research on lead adsorption to biogenic manganese

oxide biofilms grown in the laboratory on glass slides reported manganese concentrations

of 15 – 20 umol Mn/m2 (Nelson and Lion, 1996). In another experiment biogenic

manganese oxide biofilms were grown on glass slides, except this time in a natural lake

known to contain manganese oxidizing bacteria. Biofilms were tested for lead adsorption

and manganese concentrations from several experiments were reported to be from 8 – 32

umol Mn/m2 (Dong et al., 2002).

Results from this research reported a maximum manganese concentration on the

column bed of 23 umol Mn/m2. This value is in the same range as the values reported in

the other experiments mentioned above. This shows that the manganese concentration on

the bed should have been high enough to adsorb lead at the concentration that was used,

74

since about the same concentration of lead was used, 2 umol/L, in all experiments, and

that there must be some other reason that the lead adsorption observed was so low.

6.2.2 Quantity of Lead Adsorbed to the Biofilms

To determine the quantity of lead that was adsorbed to oxidized manganese in the

biofilm, data from the first complete run, in which there was a noted difference between

control and sample breakthrough curves (Figure 9), was used. The total quantity of lead

adsorbed was determined by integrating over the first five hours of lead adsorption for the

column containing oxidized manganese to find total lead adsorbed and doing the same for

the column containing only L. discophora and then subtracting the two to find the total

lead that was adsorbed by the oxidized manganese only. It was calculated that .027 umol

of lead was adsorbed by the 0.26 mg of oxidized manganese in the biofilm. This means

that 5.74 mmol Pb/mol Mn was adsorbed. This number is two orders of magnitude less

than that of previous research results, which found adsorption of lead to manganese

oxidized by L. discophora to be 550 mmol Pb/mol Mn (Nelson et al., 1999b). This

shows again that perhaps low levels of oxidized manganese were not as much of a

problem as poor adsorption of lead to that oxidized manganese.

Knowing the concentration of biogenically oxidized manganese on the column

bed, it was possible to calculate the amount of lead that could theoretically be adsorbed

using Langmuir adsorption isotherm parameters determined in previous research (Nelson

et al., 1999b). To make this comparison, we can calculate the expected lead adsorption to

the manganese oxide biofilm when 90% of the lead has been removed. If 90% of the lead

in solution was adsorbed to the biofilm, 0.028 umol/L lead would be the equilibrium lead

concentration in equilibrium with the lead adsorbed to the manganese oxide in the

75

column bed. Using this number in the Langmuir adsorption isotherm it was determined

that 349 umol of lead would be adsorbed per mol of manganese present on the column

bed. Using the maximum amount of manganese present on the bed discussed above of 23

umol Mn/m2 it was then determined that 2.54 umol of lead could be adsorbed to the

biofilm. With lead being introduced at a rate of 0.28 umol every 30 minutes, this would

mean that it would be 4.5 hours before the column become completely saturated. In

contrast, for the columns used in the 8/17/2004 experiments, the column with manganese

oxide reached the point of 10% remaining lead in solution at 2.5 hours (Figure 9), which

is about half the time expected based on equilibrium adsorption isotherms of previous

studies. The column without manganese reached this point in only about 1 hour. It

should also be noted that a lag time of up to an hour may be present in the breakthrough

curves because the columns were full of MMS media and it took some time for the lead

solution to flow through the columns. This means the effluent lead concentration may

have passed 10% in as little as 1.5 hours compared to the 4.5 hours expected.

6.3 Mass Transfer Analysis

A mass transfer analysis of the packed-bed bioreactor was performed to determine

if the lead solution had a sufficient amount of contact time with the biogenic manganese

oxide biofilm on the surface of the beads in the bed. Methods from the textbook

Transport Processes and Separation Process Principles were used for the calculation of

mass transfer rates in a packed bed (Geankoplis, 2003).

To begin this calculation a diffusion coefficient for lead in water of 1.5×10-5

cm2/sec was used. Calculations were made using a column bed height of 20 cm, a

column bed diameter of 5.5 cm, a bead diameter of 0.635 cm, a porosity of 0.295, a

76

temperature of 25 oC, a feed rate of 5 mL/min, a feed lead concentration of 2 umol/L. A

Reynold’s Number of 2.2, and a Schmidt Number of 580 were calculated. Using

Equation 3 and Equation 4, a mass transfer coefficient of 0.0066 cm/min was found. In

these equations, JD is the mass-transfer coefficient, NRe is the Reynold’s Number, ε is the

void fraction, Kc’ is the flux coefficient, V’ is the velocity, and NSe is the Schmidt

Number.

3/2

Re09.1 −= NJ D ε

(Eq. 3)

3/2'

'

)( Sec

D NVK

J = (Eq. 4)

This calculation resulted in an effluent concentration of lead of 0.031 umol/L

based on the assumption that all lead was adsorbed instantly at the surface of the beads.

For this analysis the concentration of lead at the surface of the beads was zero, because of

the assumption of instantaneous adsorption. It was also assumed that the biogenic

manganese oxide biofilms were completely and evenly covering the surface of the

column beds. This shows that under these conditions mass transfer is not a problem in

the adsorption of lead to the biogenic manganese oxide biofilm and that much higher

adsorption should have been possible up until saturation was achieved after several hours,

as discussed in the previous section. While both of the assumptions are not correct, the

calculation still shows that under this ideal scenario, mass transfer limitations in the

column bed would not be a problem and would not account for the high effluent lead

concentrations that were observed.

77

The calculations suggest that the kinetics of lead adsorption to the manganese

oxide surfaces in the packed-bed bioreactor may be an important limitation. It is highly

likely that lead is not adsorbed instantaneously or even over a short time to the biogenic

manganese oxide biofilms, but rather needs a longer amount of time to completely

adsorb. In other research, at least 24 hours were given for lead adsorption measurements

(Nelson et al, 1999b). There currently hasn’t been any research reported on the kinetics

of lead adsorption to biogenic manganese oxide biofilms and this would need to be done

to determine if indeed it is a kinetics problem with the packed-bed biofilm bioreactor that

is causing such low levels of lead adsorption.

6.4 Possible Future Experiments

Assuming that kinetics is the major problem in the experiment there are several

changes that could be made to increase the performance of a packed-bed biogenic

manganese oxide adsorption column. If a circulating solution of lead was left to adsorb

for a longer period of time it is possible that much more complete lead adsorption would

occur. Another option would be to slow down the influent flow rate of the lead solution

in order to give plenty of time for adsorption. Perhaps, if the retention time was slowed

from 30 minutes to 12 hours or even 24 hours then much more complete adsorption

would be allowed take place. A much larger or taller column, perhaps 4 or 5 ft. in length,

could also increase success. Of course, increased biofilm growth of pure L. discophora

and in turn increased levels of biogenically oxidized manganese in the column bed will

increase the potential for lead adsorption. A more densely packed column bed with

smaller beads may also help with the biofilm growth by allowing a more evenly wetted

bed. This would greatly increase the surface area available for lead adsorption. Still,

78

many of the suggestions discussed above are speculation and it would be very helpful if

straight kinetic experiments were performed on adsorption rates of lead to biogenic

manganese oxide biofilms.

6.5 Potential Applications

Once the proper changes were made to the bioreactor apparatus and high levels of

lead adsorption were achieved, it would then be possible to use that design in several

potential applications. One major application would be in filtering toxic trace metals like

the lead tested in this experiment out of wastewater. There is also the possibility of toxic

trace metal contamination to drinking water supplies, which could be very dangerous to

large populations of people. Whether the contamination was to occur naturally or by

some human intervention having a way to simply and quickly remove the contamination

from such a water supply would be very important.

Perhaps toxic materials other than trace metals could also be effectively removed

from liquid solutions by biogenic manganese oxides. One possibility of this would be to

use the biofilm bioreactor to oxidize hydrocarbons or other organic contaminants, which

are a major source of contamination in groundwater and other natural aquatic

environments. Preliminary research has shown that manganese oxides can catalyze the

oxidation of humic acids (Sunda et al., 1994), and this is an indication that they could

oxidize other recalcitrant compounds. Further research should thus be done to perfect the

apparatus for lead adsorption as well as further testing on other toxic trace metals and

toxic substances.

79

CHAPTER 7

CONCLUSIONS

7.1 Summary

Three complete runs of lead adsorption experiments were performed in packed-

bed columns containing L. discophora manganese oxide biofilms. The first complete run

showed very little microbial contamination in the columns and, results from lead

adsorption showed a difference in the adsorption of lead to the biofilm containing

oxidized manganese compared to the control with no manganese. In the second complete

run there were high levels of microbial contamination present in both columns, and no

measurable biofilm growth was observed. Results from lead adsorption showed no

significant lead adsorption and no significant difference between control and sample

columns as would be expected if there was no biofilm on the column bed. The third

complete run showed some microbial contamination, and results from lead adsorption

showed that some lead was adsorbed to the biofilms in both columns, although less than

observed in the first run.

Several steps were taken in an attempt to determine the reason or reasons that

such low levels of lead adsorption were observed. Manganese concentrations on the

column bed surface were compared to those in other research performed in which lead

adsorption had been tested. These manganese concentrations were found to be at a

similar level, 23 umol Mn/m2 in column #1 of the third run compared to 15-20 in the

literature (Nelson et al., 1999b). Lead adsorption to that oxidized manganese was then

compared, and it was found that lead adsorption from the first complete run was two

80

orders of magnitude lower than that in the previous study by Nelson et al. (1999b). This

indicates that the oxidized manganese biofilms were not saturated with lead to nearly the

extent possible based on previously determined isotherms. Therefore, the amount of

oxidized manganese present may not be the limitation of the columns, but rather the

kinetics of adsorption. Also using data from previous research, a calculation was made to

determine when saturation of the biogenic manganese oxide biofilm would occur. It was

determined that it would take 4.5 hours for saturation to occur in the column containing

the most oxidized manganese.

A mass transfer analysis was also performed to determine if the lead solution had

a sufficient amount of contact time for diffusion of lead to the surface of the manganese

oxide biofilm to occur. This analysis showed that the effluent concentration of lead

would be 0.031 umol/L if all lead that diffused to the surface of the beads was instantly

adsorbed. This means that if there was sufficient adsorption capacity and no limitation

due to kinetics of adsorption, that an effluent concentration of 0.031 umol/L would be

possible. Since the observed effluent lead concentrations were quickly much higher than

this, it is likely that the kinetics of adsorption is the limiting process.

After ruling out the possibilities of major problems discussed above, it was

determined that the low levels of lead adsorption to the biogenic manganese oxide

biofilm were most likely due to the kinetics of lead adsorption. It is currently unknown

how long it takes for lead to adsorb to a biogenic manganese oxide biofilm and further

research would need to be performed to determine this. There are, however, several steps

that could be taken to improve the possibility of success in this experiment and some of

them are discussed in the next section.

81

7.2 Future Recommendations

The following are recommendations to improve the lead adsorption success of the L.

discophora oxidized manganese packed-bed bioreactor and other future experiments that

should be performed once those improvements are made.

• Pure kinetics experiments on how fast lead binds to biogenic manganese oxide

biofilms should be performed to better determine how to change the bioreactor

apparatus for maximum lead adsorption.

• Experiments should be conducted in which a significantly slower flow rate is used

to allow more contact time with the oxidized manganese biofilm. The flow rate

could be reduced so that a single retention time was 12 or even 24 hours.

• A much larger or longer column should be experimented with to give the lead

solution more residence time in the packed-bed column. A column as long as 1 m

may be necessary to maximize adsorption.

• A more densely packed bed in which smaller beads are used should be

experimented with. This may improve biofilm growth as well as contact time

during lead adsorption.

• Further efforts should be made to reduce contamination in the reservoir and

column by improving pure culture techniques when inoculating the bioreactors

and during biofilm growth.

82

• Experiments should be conducted to increase the biomass in the packed-bed

column and in turn maximize oxidized manganese.

• Further testing should be done on other trace metals, as well as other substances

such as hydrocarbons.

83

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